Vaccination or immunization using a prime-boost regimen

ABSTRACT

Disclosed and claimed are methods and compositions and kits for the vaccination or immunization of an animal, such as a mammal, advantageously a bovine, involving a prime-boost regimen.

RELATED APPLICATIONS/INCORPORATION BY REFERENCE

[0001] This application is a continuation-in-part of U.S. applicationSer. No. 09/766,442, filed Jan. 19, 2001 as a continuation-in-part ofU.S. application Ser. No. 09/760,574, filed Jan. 16, 2001; and, thisapplication claims priority from U.S. Provisional application Serial No.60/193,126, filed Mar. 30, 2000, and French application No. 00 00798,filed Jan. 21, 2000. Mention is also made of U.S. application Ser. Nos.09/232,468, 09/232,469, and 09/232,279, each filed Jan. 15, 1999, U.S.Pat. No. 6,376,473 and U.S. application Ser. No. 10/085,519. Each of theforegoing applications and patent, and all documents cited therein orduring their prosecution (“appln cited documents”) and all documentscited or referenced in the appln cited documents, and all documentscited or referenced herein (“herein cited documents”), and all documentscited or referenced in herein cited documents, together with anymanufacturer's instructions, descriptions, product specifications, andproduct sheets for any products mentioned herein or in any documentincorporated by reference herein, are hereby incorporated herein byreference, and may be employed in the practice of the invention.

FIELD OF THE INVENTION

[0002] The present invention relates to methods of vaccination orimmunization of bovines, advantageously such methods involving aprime-boost regimen, as well as vaccines or immunological or immunogeniccompositions, such as DNA vaccines or immunogenic or immunologicalcompositions, which can be used such methods.

BACKGROUND

[0003] Deoxyribonucleic acid (DNA) molecules have been used forvaccination (Wolf et al. Science 1990. 247. 1465-1468). This type ofvaccination induces cellular and humoral immunity after in vivotransfection of cells of the subject to be vaccinated with DNA or RNAmolecules encoding immunologically active proteins.

[0004] A DNA vaccine or immunogenic or immunological composition iscomposed of at least one plasmid which may be expressed by the cellularmachinery of the subject to be vaccinated or inoculated and of apharmaceutically acceptable vehicle or excipient. The nucleotidesequence of this plasmid encodes, inter alia, one or more immunogens,such as proteins or glycoproteins capable of inducing, in the subject tobe vaccinated or inoculated, a cellular immune response (mobilization ofthe T lymphocytes) and a humoral immune response (stimulation of theproduction of antibodies specifically directed against the immunogen)(Davis H. L. Current Opinion Biotech. 1997.8.635-640).

[0005] An immunogen or immunogens derived from a pathogen may not besufficiently effective for inducing an optimum or protective immuneresponse in the animal to be vaccinated or inoculated. Therefore it maysometimes be useful to improve the immune response.

[0006] Various routes of administration for the DNA vaccines have beenproposed (intraperitoneal, intravenous, intramuscular, subcutaneous,intradermal, mucosal, and the like). Various means of administrationhave also been proposed, for instance gold particles coated with DNA andprojected so as to penetrate into the cells of the skin of the subjectto be vaccinated (Tang et al. Nature 1992. 356. 152-154) and liquid jetinjectors which make it possible to transfect both skin cells and cellsof underlying tissues (Furth et al. Analytical Bioch. 1992. 205.365-368).

[0007] Chemical compounds have been used for the in vitro transfectionof DNA:

[0008] A/—cationic lipids.

[0009] B/—the polymers, such as for example SuperFect (molecules ofactivated dendrimers, produced by Qiagen; Xu et al. Mol. Genet. Metab.1998. 64. 193-197), and

[0010] C/—the biochemical agents, such as for example toxins, e.g.,cholera toxins.

[0011] The cationic lipids may be divided into four subgroups.

[0012] 1) The cationic lipids containing quaternary ammonium salts, suchas, for example DOTMA (dioleoyloxypropyltrimethylammonium, produced byGibco under the name Lipofectine), DOTAP(trimethyl-2,3-(octadec-9-eneoyloxy)-1-propaneammonium; Gregoriadis etal. FEBS Letters 1997. 402. 107-110), DMRIE(N-(2-hydroxyethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propaneammonium;WO-A-9634109), DLRIE(N-(2-hydroxyethyl)-N,N-dimethyl-2,3-bis(dodecyloxy)-1-propaneammonium;Feigner et al. Ann. N Y Acad. Sci. 1995. 772. 126-139).

[0013] These cationic lipids containing quaternary ammonium salts may becombined or otherwise with an additional neutral lipid, such as DOPC(dioleoylphosphatidylcholine) or DOPE (dioleoylphosphatidylethanolamine)(J. P. Behr, Bioconjugate Chemistry 1994. 5. 382-389).

[0014] 2) The lipoamines, such as for example DOGS(dioctadecylamidoglycylspermine, produced by Promega under the nameTransfectam; Abdallah et al. Biol. Cell. 1995. 85. 1-7), DC-Chol(dimethylaminoethane-carbamoyl-cholesterol; Gao and Huang, Biochem.Biophys. Res. Commun. 1991. 179. 280-285), BGSC(bis-guanidine-spermidine-cholesterol), BGTC(bis-guanidine-trencholesterol) (Vigneron et al. Proc. Natl. Acad. Sci.USA 1996. 93. 9682-9686).

[0015] 3) The cationic lipids containing quaternary ammonium salts andlipoamines, such as for example DOSPA(N,N-dimethyl-N-(2-(sperminecarboxamido)ethyl)-2,3-bis(dioleoyloxy)-1-propaneimidiumpentahydrochloride, marketed by Gibco under the name LipofectAmine®;Hawley-Nelson et al. Focus 1993. 15. 73-79), GAP-DLRIE(N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(dodecyloxy)-1-propaneammonium;Wheeler et al. Proc. Natl. Acad. Sci. USA 1996. 93. 11454-11459; Normanet al. Vaccine 1997. 15. 801-803). And, 4) The lipids containing amidinesalts, such as for example ADPDE, ADODE (Ruysschaert et al. Biochem.Biophys. Res. Commun. 1994. 203. 1622-1628).

[0016] Some of these compounds have been used in the formulation of DNAvaccines with more than mitigated results. Knowledge in the field of invitro transfection is not transposable to DNA vaccination, where theobjective is to ensure an optimal, and advantageously protective, immuneresponse. Negative effects on the induction of an effective immuneresponse, e.g., protective immune response, have even been observed withcompounds known to promote transfection in vitro. And, some chemicalcompounds are toxic at high doses to the transfected cells.

[0017] In the work by Etchart (Etchart et al. J. Gen. Virol. 1997. 78.1577-1580), the use of DOTAP did not have an adjuvant effect during theadministration of the DNA vaccine by the intranasal route, whereas ithad an adjuvant effect by the oral route. DOTAP has also been used inDNA vaccines encoding the influenza virus hemagglutinin (HA) on themouse model which were administered by the intranasal route (Ban et al.Vaccine 1997. 15. 811-813), but the addition of DOTAP inhibited theimmune response. The use of DC-Chol or DOTAP/DOPE in DNA vaccinesencoding the hepatitis B virus surface protein (S) in mice which wereadministered by the intramuscular route made it possible to increase theantibody response, whereas the use of Lipofectine (or DOTMA) did notincrease this response (Gregoriadis et al. FEBS Letters 1997. 402.107-110). DC-Chol/DOPE has also been used in DNA vaccines against thehuman immunodeficiency virus (HIV, Env protein) in mice, withadministration by the intramuscular route inducing a more effectiveimmune response, whereas administration by the subcutaneous orintradermal route did not increase it (Ishii et al. AIDS Res. Hum.Retro. 1997. 13. 1421-1428). Clearly, many factors, including the routeof administration, play into whether a compound is effective inincreasing the immune respone.

[0018] The addition of certain cytokines, such as interleukins orinterferons, can make it possible to enhance the immune response inducedby DNA vaccines. Each cytokine triggers a reaction which iscytokine-specific and orients the immune response to a greater or lesserdegree towards a cellular response or towards a humoral response(Pasquini et al. Immunol. Cell. Biol. 1997. 75. 397-401; Kim et al. J.Interferon Cytokine Res. 1999. 19. 77-84). The adjuvant effects of acytokine obtained from a given species are not necessarily the same ifthe immune context varies; for instance, if a cytokine of one species isadministered to another species, e.g., in a heterologous immune system.The addition of cytokine may also have no adjuvant effect, or may evenresult in a reversal of the effect sought, that is to say a reduction oran inhibition of the immune response. Thus, a DNA vaccine encoding asingle chain of an immunoglobulin fused with GM-CSF does not increasethe immune response, whereas direct administration of this fusionprotein to mice is effective, in the same way as is the administrationof a fusion protein consisting of Fv and of the cytokine IL-1 beta orthe administration of a DNA vaccine encoding the latter fusion protein(Hakim et al. J. Immunol. 1996. 157. 5503-5511). The use of plasmidsco-expressing the cytokine IL-2 and the hepatitis B virus envelopeprotein in a fused or non-fused conformation results in an increase inthe humoral and cellular immune responses (Chow et al. J. Virol. 1997.71. 169-78). However, the use of a bicistronic plasmid encoding thehuman acquired immunodeficiency virus (HIV-1) glycoprotein gp120 and thecytokine IL-2 induced a lower specific anti-gp120 immune response thanthat obtained by the use of a monocistronic plasmid encoding only gp120(Barouch et al. J. Immunol 1998. 161. 1875-1882). The co-injection, intomice, of two expression vectors, one coding for the rabies virus Gglycoprotein, the other for murine GM-CSF stimulates the activity of theB and T lymphocytes, whereas the co-injection with a plasmid encodinggamma-interferon (in place of murine GM-CSF) results in a decrease inthe immune response (Xiang et al. Immunity 1995. 2. 129-135). Thus,whether a cytokine inhances an immune response depends on variousfactors.

[0019] Certain modifications in the antigens, such as deletions of partof the nucleotide sequence encoding the antigen, insertions of a DNAfragment into the nucleotide sequence encoding the antigen or intonon-translated regions upstream or downstream, can also enhance theefficacy of DNA vaccines, for instance by enhancing the level ofexpression of the antigen or its presentation.

[0020] However, in practice, manipulations on the nucleotide sequencesencoding the antigen may bring about a reduction or loss of the initialimmunological activity. Thus, the deletion of the transmembrane domainfrom the gene encoding the rabies virus G antigen reduced the level ofprotection induced in the mouse model after administration by theintramuscular route of a DNA vaccine encoding this modified antigen(Xiang et al. Virol. 1995. 209. 569). The deletion of the transmembranedomain from the gene encoding the bovine herpesvirus (BHV) gDglycoprotein did not make it possible to increase the antibody responseand induced only a partial protection in bovines vaccinated by theintramuscular route (van Drunen Little-van den Hurk et al. J. Gen.Virol. 1998. 79. 831-839). The humoral and cellular immune responses andthe protection conferred are identical in guinea pigs challenged afterhaving been immunized with the aid of either a DNA vaccine encoding theEbola virus GP glycoprotein, or of a DNA vaccine encoding this GPglycoprotein but in a secreted form (Xu et al. Nature Medicine 1998. 4.37-42).

[0021] The insertion of the signal sequence of the human tissueplasminogen activator (tPA) into the gene encoding the malaria Pf332antigen did not make it possible to increase the antibody response inmice vaccinated by the intramuscular route (Haddad et al. FEMS1997.18.193-202). The addition, in phase, of a tPA sequence to the geneencoding the murine rotavirus VP7 antigen also did not make it possibleto increase the antibody response in mice vaccinated by the intradermalroute, whereas the fusion protein consisting of the VP4 antigen and tPAallowed this increase, but without inducing an effective protection(Choi et al. Virology 1998. 250. 230-240).

[0022] Accordingly, whether a modification to a nucleotide sequence willbe useful depends on many factors, and modifications carried out on thenucleotide sequence of one antigen cannot in general be directlytransposed to another antigen, because antigens do not always have thesame structural arrangements.

[0023] Moreover, it would be desirable to enhance or improve vaccinationor immunization methods, for instance, the vaccination or immunizationof bovines; and, it would be desirable to provide vaccination orimmunization methods methods involving a prime-boost regimen, as well asvaccines or immunological or immunogenic compositions, such as DNAvaccines or immunogenic or immunological compositions, which can be usedsuch methods.

OBJECTS AND SUMMARY OF THE INVENTION

[0024] It has been found that DNA vaccination or immunization of animalswhich can be given DNA vaccination or immunization, e.g., mammals,avians, reptiles, advantageously bovines (e.g., cows, calves, bulls,cattle, buffalo “beefalo” and the like), can be improved by avaccination or immunization regimen; for instance, administering one ormore DNA vaccines or immunological or immunogenic compositions as a“prime” and thereafter administering one or more subunit (e.g.,antigen(s), immunogen(s) or epitope(s) preparation(s)—“subunit(s)” ofthe pathogen), and/or inactivated pathogen and/or attenuated pathogenvaccine or immunological or immunogenic compositions and/or arecombinant or modified vector, e.g., virus, bacterial or yeast, vaccineor immunogenic or immunological compositions (which contain an in vivoexpression vector, e.g., a modified or recombinant virus, bacteria,yeast or other expression vector).

[0025] The prime-boost regimen according to the invention can be used inanimals of any age, advantageously young animals (e.g., animals thathave detectable maternal antibodies and/or are suckling or nursing orbreast-feeding, such as a young calve—a calve that has detectablematernal antibodies and/or is suckling or nursing or breast-feeding),pre-adult animals (animals that are older than being a young animal buthave not yet reached maturity or adulthood or an age to mate orreproduce), adult animals (e.g., animals that are of an age to mate orreproduce or are beyond such a period in life), and it is advantageousto employ the prime-boost regimen in pregnant females or females priorto giving birth or insemination.

[0026] The prime-boost regimen is especially advantageous to practice ina young animal, e.g., a young bovine or calve, as it allowsvaccinatation or immunization at an early age, for instance, the firstadministration in the prime-boost regimen or the prime can beadministered to a young animal can be at an age at which the the younganimal has maternal antibodies. Another advantage of this regimen isthat it can provide a degree of safety for pregnant females, e.g., cows,present in the same location or in close proximity to the young or toeach other, e.g. at the same farm or that share common grazing area.

[0027] Thus, the invention provides a prime-boost immunization orvaccination method advantageously practiced in bovines against one ormore pathogens of bovines, and the method may be practiced upon a younganimal, such as a young calve, for instance, wherein the priming is doneat a time that the young animal has maternal antibodies against thebovine pathogen, with the boost advantageously at a time when maternalantibodies may be waning or decreasing or normally not present, such asduring a period of time post-breastfeeding.

[0028] The bovine pathogen against which the prime-boost regimen can beemployed includes: bovine respiratory syncitial virus (BRSV), bovineparainfluenza virus type 3 (bPI-3), bovine herpesvirus type 1 (BHV-1)(responsible for infectious bovine rhinotracheitis (IBR)), mucosaldisease virus and bovine pestivirus type 1 or type 2 (bovine viraldiarrhea virus or BVDV-1 and BVDV-2)). Advantageously, the prime-boostregimen of the invention is practiced against BRSV.

[0029] The invention further comprehends the compositions and kitsincluding one or more DNA vaccines or immunogenic or immunologicalcompositions which may be used in the prime-boost regimen of theinvention, and which make it possible to obtain an improved oradvantageously effective and/or protective immune protection in cattle,comprising at least one valency selected from the group consisting ofthe BRSV, bPI-3, BHV-1 and BVDV (comprising at least one plasmid thatcontains and expresses a nucleic acid molecule encoding at least oneimmunogen, antigen or epitope of BRSV, bPI-3, BHV-1 or BVDV).

[0030] Accordingly, the invention also involves kits for performing aprime-boost regimen comprising or consisting essentially of a primingvaccine or immunological or immunogenic composition and a boost vaccineor immunological or immunogenic compositions, in separate containers,optionally with instructions for admixture and/or administration.

[0031] The invention provides a prime-boost immunization or vaccinationmethod of a bovine (e.g., cow, bull, calve) against at least one bovinepathogen comprising administering to the bovine a priming DNA vaccine orimmunological or immunogenic composition comprising nucleic acidmolecule(s) encoding and expressing in vivo an immunogen(s), antigen(s)or epitope(s) from the pathogen, and thereafter administering a boostingvaccine or immunogenic or immunological composition that presents to thebovine's immune system the same immunogen, antigen or epitope. Theboosting vaccine or immunogenic or immunological composition isadvantageously different than the DNA vaccine or immunogenic orimmunological composition. For instance, the boosting vaccine orimmunogenic or immunological composition can be an inactivated pathogenand/or an attenuated pathogen and/or a subunit (advantageously theantigen, immunogen and/or epitope expressed by the DNA vaccine orimmunogenic or immunological composition) and/or a recombinant ormodified vector, e.g., virus, vaccine or immunogenic or immunologicalcomposition. A recombinant or modified vector is advantageously an invivo expression vector, such as a modified or recombinant bacteria,yeast, virus, e.g. poxvirus, adenovirus, herpesvirus, comprising nucleicacid molecule(s) encoding and expressing in vivo the immunogen(s),antigen(s) or epitope(s) from the pathogen expressed by the DNA vaccineor immunogenic or immunological composition. The boost is advantageouslyperformed with an inactivated vaccine or immunogenic or immunologicalcomposition, or with a vaccine or immunogenic or immunologicalcomposition comprising a recombinant live viral vector, such as arecombinant poxvirus, that comprises nucleic acid molecule(s) encodingand express(es) in vivo the immunogen(s), antigen(s) or epitope(s) fromthe pathogen expressed by the DNA vaccine or immunogenic orimmunological composition. Thus, it is advantageous that the boosteither comprises the immunogen, antigen or epitope expressed by the DNAvaccine or immunogenic or immunological composition or expresses in vivothe same immunogen, antigen or epitope expressed by the DNA vaccine orimmunogenic or immunological composition.

[0032] The terms “immunogenic composition” and “immunologicalcomposition” and “immunogenic or immunological composition” cover anycomposition that elicits an immune response against the targetedpathogen; for instance, after administration or injection into thebovine, elicits an immune response against the targeted pathogen. Theterms “vaccinal composition” and “vaccine” and “vaccine composition”covers any composition that induces a protective immune response againstthe targeted pathogen or which efficaciously protects against thepathogen; for instance, after administration or injection into thebovine, elicits a protective immune response against the targetedpathogen or provides efficacious protection against the pathogen.Furthermore, while the text speaks of “immunogen, antigen or epitope”,an immunogen can be an antigen or an epitope of an antigen.

[0033] The term of “prime-boost” refers to the successiveadministrations of two different types of vaccine or immunogenic orimmunological compositions having at least one immunogen, antigen orepitope in common. The priming administration (priming) is theadministration of a first vaccine or immunogenic or immunologicalcomposition type and may comprise one, two or more administrations. Theboost administration is the administration of a second vaccine orimmunogenic or immunological composition type and may comprise one, twoor more administrations, and, for instance, may comprise or consistessentially of annual administrations.

[0034] Thus, the invention comprehends administering to a bovine apriming composition comprising a DNA vaccine or immunogenic orimmunological composition against a bovine pathogen comprising at leastone plasmid that contains and expresses in a bovine host cell anucleotide sequence encoding an immunogen, antigen or epitope of thebovine pathogen, and thereafter a boosting composition that comprisesthe bovine pathogen as an inactivated pathogen, or the bovine pathogenas an attenuated pathogen, or the immunogen, antigen or epitopeexpressed by the DNA vaccine or immunogenic or immunologicalcomposition, or a recombinant or modified vector, e.g., virus, such as arecombinant or modified herpesvirus, adenovirus or poxvirus(advantageously poxvirus, such as a vaccinia, canarypox or fowlpoxvirus) that contains and expresses in a bovine host cell a nucleotidesequence encoding the immunogen, antigen or epitope of the bovinepathogen expressed by the DNA vaccine or immunogenic or immunologicalcomposition. The bovine pathogen can be BRSV, bPI-3, BHV-1 or BVDV.

[0035] The DNA vaccine or immunogenic composition can contain a cationiclipid containing a quaternary ammonium salt, of the formula

[0036] in which R₁ is a saturated or unsaturated linear aliphaticradical having 12 to 18 carbon atoms, R₂ is an aliphatic radicalcontaining 2 or 3 carbon atoms, and X a hydroxyl or amine group.Advantageously, this compound is DMRIE(N-(2-hydroxyethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propanammonium).This compound can be combined with a neutral lipid, such as, DOPE(dioleoyl-phosphatidyl-ethanolamine). When the compound is DMRIE and theneutral lipid is DOPE, and they are combined, they form DMRIE-DOPE.

[0037] Alternatively or additionally, the DNA vaccine or immunogeniccomposition can contain bovine GM-CSF, or an expression vector thatcontains and expresses in a bovine host cell a nucleotide sequenceencoding bovine GM-CSF. The expression vector that contains andexpresses the bovine GM-CSF can be a plasmid, or a recombinant ormodified vector such as a recombinant or modified virus, bacteria,yeast. The plasmid in the DNA vaccine or immunogenic composition thatexpresses the immunogen, antigen or epitope of the bovine pathogen canalso express bovine GM-CSF.

[0038] Even further alternatively or additionally, in the DNA vaccine orimmunogenic composition, the nucleotide sequence encoding the immunogen,antigen or epitope of the bovine pathogen can have deleted therefrom aportion encoding a transmembrane domain. Yet even further alternativelyor additionally, the plasmid in the DNA vaccine or immunogeniccomposition can further contain and express in a bovine host cell anucleotide sequence encoding a heterologous tPA signal sequence such ashuman tPA and/or a stabilizing intron, such as intron II of the rabbitbeta-globin gene.

[0039] Advantageously, the bovine pathogen is BRSV; for example, theimmunogen can be BRSV F or G or N. Even more advantageously, theimmunogen is BRSV F or G, modified by substitution of the BRSV F and/orG signal sequence with a human tPA signal sequence, and/or by deletionof the transmembrane domain and/or cytoplasmic tail. The coding for theF protein can also contain a deletion of nucleotides upstream from thetransmembrane domain and corresponding to 1 and up to 92 amino acids.

[0040] Thus, the DNA vaccine or immunogenic composition can comprise afirst plasmid that contains and expresses in a bovine host cell anucleotide sequence encoding BRSV F, modified by substitution of theBRSV F signal sequence with a human tPA signal sequence and deletion ofthe transmembrane domain and contiguous C-terminal portion (cytoplasmictail) optionally also with deletion of the upstream region describedabove; and a second plasmid that contains and expresses in a bovine hostcell a nucleotide sequence encoding BRSV N and/or BRSV G, modified bysubstitution of the BRSV G signal sequence with a human tPA signalsequence and deletion of the transmembrane domain and contiguousC-terminal portion (cytoplasmic tail); and wherein the cationic lipid ispresent, e.g., DMRIE, and the neutral lipid is also present, e.g., DOPE,for instance whereby the vaccine or immunogenic composition comprisesDMRIE-DOPE. This DNA vaccine or immunogenic composition can furthercomprise bovine GM-CSF or an expression vector (e.g., plasmid) thatcontains and expresses in a bovine host cell a nucleotide sequenceencoding bovine GM-CSF. Additionally or alternatively, the DNA vaccineor immunogenic composition can contain a plasmid that expresses all ofthese “optimised” BRSV immunogens, and advantageously further containsthe cationic lipid is present, e.g., DMRIE, and the neutral lipid ispresent, e.g., DOPE, for instance, whereby the DNA vaccine orimmunogenic composition comprises DMRIE-DOPE; and, this DNA vaccine orimmunogenic composition can further comprise bovine GM-CSF or anexpression vector (e.g., plasmid) that contains and expresses in abovine host cell a nucleotide sequence encoding bovine GM-CSF.

[0041] In the practice of the invention, the bovine pathogen can bebovine pathogen is BHV-1, and the immunogen can be BHV-1 gB and/or BHV-1gC and/or BHV-1 gD. The immunogen can be BHV-1 gB, modified bysubstitution of the BHV-1 gB signal sequence with a human tPA signalsequence, and/or by deletion of the transmembrane domain and/or BHV-1gC, modified by substitution of the BHV-1 IgC signal sequence with ahuman tPA signal sequence, and/or by deletion of the transmembranedomain and/or BHV-1 gD, modified by substitution of the BHV-1 gD signalsequence with a human tPA signal sequence, and/or by deletion of thetransmembrane domain.

[0042] Thus, the DNA vaccine or immunogenic composition can comprise afirst plasmid that contains and expresses in a bovine host cell anucleotide sequence encoding BHV-1 gB, modified by substitution of theBHV-1 gB signal sequence with a human tPA signal sequence and deletionof the transmembrane domain and contiguous C-terminal portion; a secondplasmid that contains and expresses in a bovine host cell a nucleotidesequence encoding BHV-1 gC, modified by substitution of the BHV-1 gCsignal sequence with a human tPA signal sequence and deletion of thetransmembrane domain and contiguous C-terminal portion; a third plasmidthat contains and expresses in a bovine host cell a nucleotide sequenceencoding BHV-1 gD, modified by substitution of the BHV-1 gD signalsequence with a human tPA signal sequence and deletion of thetransmembrane domain and contiguous C-terminal portion; and wherein thecationic lipid is present, e.g., DMRIE, and the neutral lipid ispresent, e.g., DOPE, for instance, whereby the DNA vaccine orimmunogenic composition comprises DMRIE-DOPE. This DNA vaccine orimmunogenic composition can further comprise bovine GM-CSF or anexpression vector (e.g., plasmid) that contains and expresses in abovine host cell a nucleotide sequence encoding bovine GM-CSF.Additionally or alternatively, the DNA vaccine or immunogeniccomposition can contain a plasmid that expresses all of these“optimised” BHV-1 immunogens, and advantageously further contains thecationic lipid is present, e.g., DMRIE, and the neutral lipid ispresent, e.g., DOPE, for instance, whereby the DNA vaccine orimmunogenic composition comprises DMRIE-DOPE; and, this DNA vaccine orimmunogenic composition can further comprise bovine GM-CSF or anexpression vector (e.g., plasmid) that contains and expresses in abovine host cell a nucleotide sequence encoding bovine GM-CSF.

[0043] In the practice of the invention the bovine pathogen can be BVDV,and the immunogen the E0 protein (gp48) and/or the E2 protein (gp53)Thus, the DNA vaccine or immunogenic or immunological composition cancomprise the nucleotide sequence(s) encoding BVDV E0 and/or E2 proteins.The immunogen can be BVDV E0, modified by being encoded by a nucleicacid into which has been inserted coding for a signal sequence, e.g., ahuman tPA signal sequence, and/or into which has been inserted an intronsuch as intron II of rabbit beta-globin, and/or BVDV E2 modified bybeing encoded by a nucleic acid into which has been inserted coding fora signal sequence, e.g., a human tPA signal sequence, and/or which hashad deleted therefrom coding for the transmembrane domain of E2 and/orthe cytoplasmic tail and/or into which has been inserted an intron suchas intron II of rabbit beta-globin.

[0044] Thus, the DNA vaccine or immunogenic composition can comprise afirst plasmid that contains and expresses in a bovine host cell anucleotide sequence encoding BVDV E0, modified by containing a human tPAsignal sequence and intron II of rabbit beta-globin; and a secondplasmid that contains and expresses in a bovine host cell a nucleotidesequence encoding BVDV E2, modified containing a human tPA signalsequence and intron II of rabbit beta-globin and deletion of thetransmembrane domain and cytoplasmic tail; and wherein the cationiclipid is present, e.g., DMRIE, and the neutral lipid is present, e.g.,DOPE, for instance, whereby the DNA vaccine or immunogenic compositioncomprises DMRIE-DOPE. This DNA vaccine or immunogenic composition canfurther comprise bovine GM-CSF or an expression vector (e.g., plasmid)that contains and expresses in a bovine host cell a nucleotide sequenceencoding bovine GM-CSF. Additionally or alternatively, the DNA vaccineor immunogenic composition can contain a plasmid that expresses all ofthese “optimised” BVDV immunogens, and advantageously further containsthe cationic lipid is present, e.g., DMRIE, and the neutral lipid ispresent, e.g., DOPE, for instance, whereby the DNA vaccine orimmunogenic composition comprises DMRIE-DOPE; and, this DNA vaccine orimmunogenic composition can further comprise bovine GM-CSF or anexpression vector (e.g., plasmid) that contains and expresses in abovine host cell a nucleotide sequence encoding bovine GM-CSF. As thereare different types of BVDV, namely BVDV-1 and BVDV-2, the E0 and E2 canbe from either or both of the BVDV types. Thus, BVDV DNA vaccines orimmunogenic compositions of the invention can contain a plasmid orplasmids containing and expressing nucleic acid molecules of BVDV-1, orBVDV-2, or both BVDV-1 and BVDV-2. Accordingly, the foregoing “twoplasmid” composition can be a “four plasmid” composition to address bothtypes of BVDV. Thus, the invention comprehends a mixture of plasmids.The mixture may comprise at least two expression plasmids, eachexpressing a different immunogen (E0 or E2) and/or obtained from adifferent type of BVDV (BVDV-1 or BVDV-2), such as a mixture made offour plasmids expressing BVDV-1 E0, BVDV-1 E2, BVDV-2 E0 and BVDV-2 E2.

[0045] In the practice of the invention, the bovine pathogen can bebPI-3; for example, the immunogen can be bPI-3 F or HN. Even moreadvantageously, the immunogen is bPI-3 F or HN, modified by substitutionof the bPI-3F and/or HN signal sequence with a human tPA signalsequence, and/or by deletion of the transmembrane domain and/orcytoplasmic tail, and/or by insertion into the nucleic acid moleculecoding therefor of an intron, such as intron II of rabbit beta-globin.

[0046] Thus, the DNA vaccine or immunogenic composition can comprise afirst plasmid that contains and expresses in a bovine host cell anucleotide sequence encoding bPI-3 F, modified by insertion of intron IIof rabbit beta-globin, substitution of the bPI-3 F signal sequence witha human tPA signal sequence and deletion of the transmembrane domain andcytoplasmic tail; and a second plasmid that contains and expresses in abovine host cell a nucleotide sequence encoding bPI-3 HN, modified byinsertion of intron II of rabbit beta-globin, substitution of the bPI-3HN signal sequence with a human tPA signal sequence and deletion of thetransmembrane domain and cytoplasmic tail; and wherein the cationiclipid is present, e.g., DMRIE, and the neutral lipid is also present,e.g., DOPE, for instance whereby the vaccine or immunogenic compositioncomprises DMRIE-DOPE. This DNA vaccine or immunogenic composition canfurther comprise bovine GM-CSF or an expression vector (e.g., plasmid)that contains and expresses in a bovine host cell a nucleotide sequenceencoding bovine GM-CSF. Additionally or alternatively, the DNA vaccineor immunogenic composition can contain a plasmid that expresses all ofthese “optimised” bPI-3 immunogens, and advantageously further containsthe cationic lipid is present, e.g., DMRIE, and the neutral lipid ispresent, e.g., DOPE, for instance, whereby the DNA vaccine orimmunogenic composition comprises DMRIE-DOPE; and, this DNA vaccine orimmunogenic composition can further comprise bovine GM-CSF or anexpression vector (e.g., plasmid) that contains and expresses in abovine host cell a nucleotide sequence encoding bovine GM-CSF.

[0047] And, in general, DNA plasmids for DNA vaccines or immunogenic orimmunological compositions, and DNA vaccines or immunogenic orimmunological compositions employed in the “priming” of the hereinprime-boost method may be as in U.S. Pat. No. 6,376,473 and U.S.applications Ser. Nos. 10/085,519, 09/766,442, 09/760,574, 60/193,126,09/232,468, 09/232,469, and 09/232,279, and French application No. 0000798, filed Jan. 21, 2000.

[0048] These and other embodiments are disclosed or are obvious from andencompassed by, the following Detailed Description.

BRIEF DESCRIPTION OF DRAWINGS

[0049] The following Detailed Description, given by way of example, andnot intended to limit the invention to specific embodiments described,may be understood in conjunction with the accompanying Figures,incorporated herein by reference, in which:

[0050]FIG. 1 shows plasmid pVR1012;

[0051]FIG. 2 shows plasmid pAB110;

[0052]FIG. 3 shows a graph representing the evolution of the rectaltemperature after challenge according to example 11;

[0053]FIG. 4 shows a graph representing the respiratory rate afterchallenge according to example 11;

[0054]FIG. 5 shows a graph representing the clinical scores afterchallenge according to example 11;

[0055]FIG. 6 shows a graph representing the lung lesion scores afterchallenge according to example 11;

[0056]FIG. 7 shows a graph representing the viral excretion afterchallenge according to example 11; and,

[0057]FIG. 8 shows a graph representing the memory BRSV-specific IFN□+Tcell response after challenge according to example 11.

[0058] Sequence Listing:

[0059] SEQ ID NO 1: oligonucleotide PB326

[0060] SEQ ID NO 2: oligonucleotide PB329

[0061] SEQ ID NO 3: oligonucleotide SB090

[0062] SEQ ID NO 4: oligonucleotide SB091

[0063] SEQ ID NO 5: oligonucleotide LF091

[0064] SEQ ID NO 6: oligonucleotide LF002

[0065] SEQ ID NO 7: oligonucleotide PB234

[0066] SEQ ID NO 8: oligonucleotide PB235

[0067] SEQ ID NO 9: oligonucleotide PB511

[0068] SEQ ID NO 10: oligonucleotide PB512

[0069] SEQ ID NO 1: oligonucleotide SB221

[0070] SEQ ID NO 12: oligonucleotide SB222

[0071] SEQ ID NO 13: oligonucleotide PB507

[0072] SEQ ID NO 14: oligonucleotide PB508

[0073] SEQ ID NO 15: oligonucleotide PB513

[0074] SEQ ID NO 16: oligonucleotide PB514

[0075] SEQ ID NO 17: oligonucleotide SB223

[0076] SEQ ID NO 18: oligonucleotide SB224

[0077] SEQ ID NO 19: oligonucleotide PB497

[0078] SEQ ID NO 20: oligonucleotide PB498

[0079] SEQ ID NO 21: oligonucleotide SB225

[0080] SEQ ID NO 22: oligonucleotide SB226

[0081] SEQ ID NO 23: oligonucleotide SB210

[0082] SEQ ID NO 24: oligonucleotide SB211

[0083] SEQ ID NO 25: oligonucleotide SB212

[0084] SEQ ID NO 26: oligonucleotide SB220

[0085] SEQ ID NO 27: oligonucleotide SB213

[0086] SEQ ID NO 28: oligonucleotide SB214

[0087] SEQ ID NO 29: oligonucleotide SB215

[0088] SEQ ID NO 30: oligonucleotide SB216

[0089] SEQ ID NO 31: oligonucleotide LF050

[0090] SEQ ID NO 32: oligonucleotide LF151

[0091] SEQ ID NO 33: oligonucleotide LF052

[0092] SEQ ID NO 34: oligonucleotide LF053

[0093] SEQ ID NO 35: oligonucleotide LF039

[0094] SEQ ID NO 36: oligonucleotide LF034

[0095] SEQ ID NO 37: oligonucleotide LF041

[0096] SEQ ID NO 38: oligonucleotide LF042

[0097] SEQ ID NO 39: oligonucleotide LF043

[0098] SEQ ID NO 40: oligonucleotide LF044

[0099] SEQ ID NO 41: oligonucleotide LF045

[0100] SEQ ID NO 42: oligonucleotide LF046

[0101] SEQ ID NO 43: oligonucleotide LF064

[0102] SEQ ID NO 44: oligonucleotide LF065

[0103] SEQ ID NO 45: oligonucleotide LF066

[0104] SEQ ID NO 46: oligonucleotide LF067

[0105] SEQ ID NO 47: oligonucleotide LF047

[0106] SEQ ID NO 48: oligonucleotide LF048

[0107] SEQ ID NO 49: oligonucleotide LF058

[0108] SEQ ID NO 50: oligonucleotide LF059

[0109] SEQ ID NO 51: oligonucleotide LF056

[0110] SEQ ID NO 52: oligonucleotide LF061

[0111] SEQ ID NO 53: oligonucleotide LF062

[0112] SEQ ID NO 54: oligonucleotide LF063

[0113] SEQ ID NO 55: oligonucleotide LF054

[0114] SEQ ID NO 56: oligonucleotide LF055

[0115] SEQ ID NO 57: oligonucleotide FC129

[0116] SEQ ID NO 58: oligonucleotide FC130

[0117] SEQ ID NO 59: oligonucleotide FC131

DETAILED DESCRIPTION

[0118] As discussed herein, the invention involves a prime-boost methodthat is advantageously practiced in bovines, such as young calves thatcan have maternal antibodies against the pathogenic agent against whichimmunization or vaccination is directed.

[0119] The DNA vaccine or immunological or immunogenic composition canbe administered to the young animal, calve; wherein a “young animal” or“young calve” is a calve from calving up to and including 12 weeks ofage, such as from calving up to and including 6 weeks of age,advantageously from calving up to and including 4 weeks of age, e.g.,from calving up to and including 3 weeks of age.

[0120] The boost administration advantageously may be administered fromabout 2 weeks to about 5 months after the priming administration, suchas from about 3 to 6 weeks after the priming administration, andadvantageously about 4 weeks after the priming administration thepriming administration. A second administration of the boost vaccine orimmunological or immunogenic composition may occur, such as when calvesare transferred to finishing units.

[0121] The DNA vaccine or immunogenic or immunological compositioncomprises, as an active ingredient, a plasmid comprising a nucleic acidmolecule which codes for an immunogen, antigen or epitope of apathogenic agent, advantageously a bovine pathogenic agent. The termplasmid covers a DNA transcription unit comprising a polynucleotidesequence comprising the sequence of the nucleic acid molecule to beexpressed and the elements necessary for its expression in vivo. Thecircular plasmid form, supercoiled or otherwise, is within the scope ofthe invention. The linear form also falls within the scope of thisinvention.

[0122] Each plasmid comprises a promoter, for expression of the nucleicacid molecule encoding the immunogen, antigen or epitope, and thus, thenucleic acid molecule encoding the immunogen, antigen or epitope isoperably linked to or under the control of the promoter. Advantageously,the promoter is a eukaryotic promoter, even more advantageously a strongpromoter, such as a strong eukaryotic promoter, e.g., a cytomegalovirusearly promoter CMV-IE, of human or murine origin, or optionally ofanother origin such as murine, rat or guinea pig. Functionalsubfragments of these promoters, i.e., portions of these promoters thatmaintain an adequate promoting activity, are included within the presentinvention, e.g. truncated CMV-IE promoters according to WO98/00166 orU.S. Pat. No. 6,156,567 can be used in the practice of the invention. Apromoter in the practice of the invention consequently includesderivatives and subfragments of a full-length promoter that maintain anadequate promoting activity and hence function as a promoter,advantageously promoting activity substantially similar to that of theactual or full-length promoter from which the derivative or subfragmentis derived, e.g., akin to the activity of the truncated CMV-IE promotersof U.S. Pat. No. 6,156,567 to the activity of full-length CMV-IEpromoters. Thus, a CMV-IE promoter in the practice of the invention cancomprise or consist essentially of or consist of the promoter portion ofthe full-length promoter and/or the enhancer portion of the full-lengthpromoter, as well as derivatives and subfragments. More generally, thepromoter can be of viral origin or of cellular origin. As a viralpromoter other than CMV-IE, the promoter can be the SV40 virus early orlate promoter or the Rous Sarcoma virus LTR promoter. The promoter canalso be a promoter from a bovine pathogenic agent, e.g., from thepathogenic agent from which the nucleic acid molecule encoding theantigen, immunogen or epitope is derived, for example the promoterspecific to the nucleic acid molecule encoding the antigen, immunogen orepitope. A cellular promoter that can be employed in the practice of theinvention is a promoter of a cytoskeleton gene, such as, for example,the desmin promoter, or alternatively the actin promoter. When severalgenes are present in the same plasmid, they may be provided in the sametranscription unit or in several different units.

[0123] The plasmid(s) in the DNA vaccines or immunogenic orimmunological compositions are in a veterinarily acceptable vehicle orexcipient. In general, the vehicle or excipient (Remington 'sPharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton,Pa., 15th Edition, 1975) can be any usual injectable fluid such aswater, physiological saline, balanced salt solution, aqueous dextrose,glycerol or the like. Hence, the DNA vaccines or immunogenic orimmunological compositions can advantageously contain a veterinarilyacceptable vehicle or excipient.

[0124] The DNA vaccines or immunogenic or immunological compositionsaccording to the invention can aldo be adjuvanted, i.e., they cancontain an adjuvant. Examples of adjuvants include non-methylated CpGgroups (Klinman D. M. et al., Proc. Natl. Acad. Sci. USA 93:2879-2883,1996; WO 98/16247), or aluminum hydroxide, aluminum phosphate, aluminumoxide (“Vaccine Design, The subunit and adjuvant approach,”Pharmaceutical Biotechnology, vol. 6, Edited by Micheal F. Powell andMark J. Newman, 1995, Plenum Press New York). Adjuvants advantageouslyemployed in the practice of the invention are the cationic lipids, suchas those containing a quaternary ammonium salt of formula:

[0125] in which R₁ is a saturated or unsaturated linear aliphaticradical having 12 to 18 carbon atoms, R₂ is another aliphatic radicalcontaining 2 or 3 carbon atoms, and X a hydroxyl or amine group.

[0126] Advantageously, this cationic lipid is DMRIE(N-(2-hydroxyethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propanammonium;WO-A-9634109).

[0127] The cationic lipid may be combined with a neutral lipid, such as,DOPE (dioleoyl-phosphatidyl-ethanolamine). DMRIE and DOPE formDMRIE-DOPE.

[0128] Advantageously, the plasmid is mixed with the cationic lipidimmediately before use and it is even more advantageous, before itsadministration to the animal, to allow the mixture thus prepared to forma complex, for example for sitting after admixture for a period rangingfrom about 10 to about 60 minutes, such as about 30 minutes.

[0129] When the neutral lipid, e.g., DOPE, is present, the cationiclipid:neutral lipid, e.g., DMRIE:DOPE, molar ratio advantageously rangesfrom about 95:5 to about 5:95, and is even more advantageously about1:1.

[0130] The plasmid:cationic lipid, e.g., DMRIE, or cationiclipid-neutral lipid, e.g., DMRIE-DOPE, adjuvant weight ratio may rangefrom about 50:1 to about 1:10, in such as from about 10:1 to about 1:5,advantageously from about 1:1 to about 1:2.

[0131] The DNA vaccines or immunogenic or immunological compositionsaccording to the invention can be formulated with a liposome, in thepresence or not of an adjuvant as described above.

[0132] Additionally or alternatively, DNA vaccines or immunogenic orimmunological compositions of the invention contain GM-CSF (granulocytemacrophage-colony stimulating factor; Clark S.C. et al. Science 1987.230. 1229; Grant S. M. et al. Drugs 1992. 53. 516), or an expressionvector that so expresses GM-CSF, with the “expression vector” includingthe plasmid that expresses the antigen, immunogen or epitope of thebovine pathogen. Thus, to the DNA vaccine or immunogenic orimmunological composition is added GM-CSF or a vector that expressesGM-CSF, e.g. added to the non-adjuvanted or adjuvanted and/or liposomeformulated vaccines or immunogenic or immunological compositions; or,the DNA plasmid that expresses the antigen, immunogen or epitope of thebovine pathogen is constructed so that it also expresses GM-CSF. If anexpression vector is providing the GM-CSF, a nucleic acid sequenceencoding GM-CSF is in the expression vector under conditions allowingits expression in vivo (e.g., it is operably linked to s suitablepromoter). Advantageously, the expression vector that expresses theGM-CSF is a plasmid, e.g. the plasmid containing the nucleotide sequenceencoding the immunogen(s) of interest (encoding the bovine antigen,immunogen or epitope), or another plasmid. The GM-CSF is advantageouslybovine GM-CSF (Maliszewski et al., Molec. Immunol., 1988, 25, 843-850).

[0133] In the vaccines or immunogenic or immunological compositionsaccording to the invention, e.g. in the non-adjuvanted or adjuvantedand/or liposome formulated vaccines or immunogenic or immunologicalcompositions, containing or not GM-CSF or an expression vectorexpressing GM-CSF, the nucleotide sequence(s) encoding the immunogen arein an optimised or modified form. Optimization is understood to mean anymodification of the nucleotide sequence which manifests itself at leastby a higher level of expression of this nucleotide sequence, and/or byan increase in the stability of the messenger RNA encoding this antigen,and/or by the triggered secretion of this antigen into the extracellularmedium, and which may have as direct or indirect consequence an increasein the immune response induced.

[0134] In the present invention, the optimization of the nucleotidesequence(s) encoding the immunogen may consist in the deletion of thefragment of the nucleotide sequence encoding the transmembrane domain ofthe immunogen (with deletion understood to mean the complete deletion ora partial deletion or disruption sufficient for the transmembrane domainto no longer, or no longer substantially, be functional, such thatdeletion of the nucleotide sequence encoding the transmembrane domaincan include disruption of the coding sequence), and/or the addition, inframe, of a nucleotide sequence encoding a heterologous (to thepathogen) tPA signal sequence such as the human tPA signal sequence(Montgomery et al. Cell. Mol. Biol. 1997. 43. 285-292; Harris et al.Mol. Biol. Med 1986. 3. 279-292), and/or in insertion of a stabilizingintron, advantageously upstream of the nucleic acid molecule (encodingthe immunogen, antigen or epitope) to be expressed, such as intron II orrabbit beta-globin. The deletion of the DNA fragment encoding thetransmembrane domain of the antigen of interest promotes the secretion,into the extracellular medium, of the antigens thus truncated and thusincreases the likelihood of the antigens coming into contact with thecells of the immune system. The insertion of the nucleotide sequenceencoding the tPA signal facilitates the translatability of the messengerRNA to which the tPA signal is joined, and thus increases the level ofexpression of this messenger RNA and therefore the production ofantigens. The tPA signal also plays a role in the secretion of theantigen synthesized. Other nucleotide sequences encoding signal peptidesmay be used, such as those for the signal peptide of melittin obtainedfrom bees (Sisk W. P. et al., 1994, J. Virol., 68, 766-775). Theinsertion of a stabilizing intron into the nucleic acid moleculeencoding the antigen of interest avoids the aberrant splicings of itsmessenger RNA and maintains the physical integrity of the latter.

[0135] Advantageously, the tPA signal is of human origin. The nucleotidesequence of the human tPA signal is accessible from the GenBank databaseunder the accession number NM_(—)000930. Advantageously, the intron isintron II of the rabbit beta-globin gene (van Ooyen et al. Science 1979.206. 337-344), whose nucleotide sequence is accessible from the GenBankdatabase under the accession number V00882 and designated by a referenceunder intron No. 2.

[0136] In an embodiment, the present invention involves vaccination orimmunization against bovine respiratory syncytial virus (BRSV).

[0137] BRSV virus is a Paramyxovirus, also a member of theParamyxoviridae family (Baker et al., Vet. Clin. North Am. Food Anim.Pract., 1997, 13, 425-454).

[0138] Nucleotide sequences encoding the F glycoprotein, the N proteinand the G glycoprotein are known and accessible from the GenBankdatabase respectively under the accession number Y17970, M35076 andU33539. The DNA vaccine or immunogenic or immunological composition usedin the prime-boost regimen against BRSV may thus comprise the nucleotidesequence(s) encoding the F, N and/or G proteins.

[0139] The nucleotide sequences and the antigens encoded therefrom maybe modified. This can be carried out by substitution, by a “signal”sequence, such as the signal sequence of human tPA, for the signalsequence of the F protein of BRSV and/or for the G envelope glycoproteinof BRSV, and/or by the deletion of the DNA fragment encoding thetransmembrane domain of F and/or of G. The deletion of the DNA fragmentencoding the transmembrane domain of one of these proteins isadvantageously accompanied by the deletion of the cytoplasmic tail. Itis possible to increase the level of expression of the F glycoprotein byfurther deleting the nucleotide sequence upstream from the transmembranedomain and corresponding to 1 and up to 92 amino acids.

[0140] Advantageously, the DNA vaccine or immunogenic or immunologicalcomposition against BRSV comprises the nucleotide sequence encoding theF protein, or the nucleotide sequence encoding the N protein, or thenucleotide sequences encoding the F protein and the N protein.

[0141] In a particular embodiment, the DNA vaccine or immunogenic orimmunological composition comprises the nucleotide sequences encodingthe F protein, wherein the nucleotide sequence encoding the F protein ismodified. This modification is chosen from among:

[0142] i. substitution, of the “signal” sequence with a heterologoussignal sequence, such as that of the tPA of human origin,

[0143] ii. deletion of the DNA fragment encoding the transmembranedomain of F and advantageously of the cytoplasmic tail,

[0144] iii. deletion of the DNA fragment encoding the transmembranedomain, of the cytoplasmic tail and of the upstream region describedabove,

[0145] iv. substitution, of the “signal” sequence with a heterologoussignal sequence, such as that of the tPA of human origin, and deletionof the DNA fragment encoding the transmembrane domain of F andadvantageoulsy of the cytoplasmic tail, or

[0146] v. substitution, of the “signal” sequence with a heterologoussignal sequence, such as that of the tPA of human origin, and deletionof the DNA fragment encoding the transmembrane domain of F, of thecytoplasmic tail and of the upstream region described above.

[0147] In a second embodiment, the DNA vaccine or immunogenic orimmunological composition comprises the nucleotide sequences encodingthe F protein and the N protein, either one plasmid containing bothnucleotide sequences, or two separate plasmids, one containing thenucleotide sequence encoding the F protein, and one containing thenucleotide sequence encoding the N protein.

[0148] In a third embodiment, the DNA vaccine or immunogenic orimmunological composition comprises the nucleotide sequences encoding Fprotein and the N protein, either one plasmid containing both nucleotidesequences, or two separate plasmids, one containing the nucleotidesequence encoding the F protein, and one containing the nucleotidesequence encoding the N protein, wherein the nucleotide sequenceencoding the F protein is modified as described above, with themodification being chosen among those described under paragraphs i to v.

[0149] Nucleotide sequences encoding the BRSV antigens which can be usedin the present invention and various expression vector constructs aregiven herein, e.g. in the accompanying examples, and in FR-A1-2751229,such as in Examples 9 and 10, and in FIGS. 5 and 6 (see also U.S. Pat.No. 6,376,473 and U.S. application Ser. No. 10/085,519).

[0150] The DNA vaccine or immunogenic or immunological compositionagainst BRSV as described above can advantageously include aveterinarily acceptable vehicle or excipient according to theabove-discussion of gthe invention.

[0151] The DNA vaccine or immunogenic or immunological compositionagainst BRSV as described above can also comprises an adjuvant accordingto above-discussion of the invention, advantageously DMRIE, even moreadvantageously, DMRIE-DOPE.

[0152] The DNA vaccine or immunogenic or immunological compositionagainst BRSV as described above, adjuvanted or not, can comprises GM-CSFor an expression vector, advantageously a plasmid, expressing GM-CSF,with the GM-CSF being advantageously bovine GM-CSF.

[0153] The addition of bovine GM-CSF may be carried out by theincorporation of the bovine GM-CSF polypeptide into the vaccinal orimmunogenic or immunological composition or advantageously by theinsertion of the nucleotide sequence encoding the bovine GM-CSF into anin vivo expression vector, such as a plasmid. The nucleotide sequenceencoding GM-CSF can be inserted into a second expression plasmid (e.g.pLF1032 Example 8), different from that (or those) into which thegene(s) encoding the BRSV antigen(s) is(are) inserted.

[0154] A nucleotide sequence encoding bovine GM-CSF is accessible fromthe GenBank database under the accession number U22385.

[0155] Advantageously, according to the invention, the DNA vaccine orimmunogenic or immunological composition against BRSV, which may beformulated with DMRIE-DOPE, is composed of an expression plasmid (e.g.pSB108 Example 4.1.2) encoding the F antigen of BRSV optimized by thedeletion of the fragment of the nucleotide sequence of F encoding thetransmembrane domain and the cytoplasmic tail, optionally also withdeletion of the upstream region described above (e.g. pPB449 Example4.1.4), and of a second expression plasmid (e.g. pFC123 Example 4.3)encoding the native N protein of BRSV.

[0156] In another embodiment, the present invention relates tovaccination or immunization against bovine parainfluenza virus type 3(bPI-3).

[0157] The bPI-3 virus is a Paramyxovirus, also a member of theParamyxoviridae family (Tsai et al., Infect. Immun., 1975, 11, 783-803).

[0158] Nucleotide sequences encoding the hemagglutinin and neuraminidaseproteins (HN) and the fusion protein (F) of bPI-3 are known andaccessible from the GenBank database under the accession number U3 1671.The DNA vaccine or immunogenic or immunological composition againstbPI-3 may thus comprise a DNA plasmid comprising nucleotide sequence(s)encoding the HN and/or F proteins.

[0159] The DNA vaccine or immunogenic or immunological compositionagainst bPI-3 as described above advantageously contains a veterinarilyacceptable vehicle or excipient according to the above-discussion of theinvention.

[0160] The DNA vaccine or immunogenic or immunological compositionagainst bPI-3 as described above can comprise an adjuvant according tothe above-discussion of the invention, such as DMRIE, advantageouslyDMRIE-DOPE.

[0161] These embodiements may optionally further include (1) theaddition of GM-CSF or an expression vector, advantageously a plasmid,expressing GM-CSF, or (2) the optimization of at least one bPI-3antigen, or (3) the addition of GM-CSF or an expression vector,advantageously a plasmid, expressing GM-CSF and the optimization of atleast one bPI-3 antigen. The GM-CSF is advantageously bovine GM-CSF. Theaddition of GM-CSF may be carried out as is described for BRSV and inthe above-discussion of the invention.

[0162] The optimization of the antigens derived from bPI-3 is carriedout by substitution, of a “signal” sequence, for example, by thesubstitution of a bPI-3 antigen signal sequence with a heterologoussignal sequence, such as the signal sequence of human tPA, e.g., thesubstitution of the signal sequence of hemagglutinin-neuraminidase (HN)of bPI-3 and/or of the fusion protein (F) of bPI-3 with the signalsequence of human tPA, and/or by the deletion of the DNA fragmentencoding the transmembrane domain of HN and/or of F, and/or by theinsertion of an intron, such as intron II of the rabbit beta-globin,advantageously upstream of the nucleotide sequence encoding HN and/or F.The deletion of the DNA fragment encoding the transmembrane domain ofone of these proteins is advantageously accompanied by deletion of thecytoplasmic tail. The DNA vaccine or immunogenic or immunologicalcomposition against bPI-3 according to the invention may thereforeencode and express a single optimized PI-3 antigen (HN or F) or both (HNand F).

[0163] Nucleotide sequences encoding the bPI-3 antigens which can beused in the present invention and various expression vector constructsare given herein, e.g. the accompanying examples, and in FR-A1-2751229,such as in Examples 14 and 15, and in FIGS. 10 and 11 (see also U.S.Pat. No. 6,376,473 and U.S. application Ser. No. 10/085,519).

[0164] Advantageously, according to the invention, the DNA vaccine orimmunogenic or immunological composition against bPI-3 comprisesDMRIE-DOPE, and is composed of an expression plasmid (e.g. pLF1025Example 7.1.2) encoding the HN antigen of bPI-3 optimized by theinsertion of the signal sequence of the human tPA in place of the signalsequence of HN, by the deletion of the fragment of the nucleotidesequence of HN encoding the transmembrane domain and the cytoplasmictail and by the insertion of intron II of the rabbit beta-globin geneupstream of HN, and of a second expression plasmid (e.g. pLF1027 Example7.2.2) encoding the F antigen of bPI-3 optimized by the insertion of thesignal sequence of the human tPA in place of the signal sequence of F,by the deletion of the fragment of the nucleotide sequence encoding thetransmembrane domain of F and the cytoplasmic tail and by the insertionof intron II of the rabbit beta-globin gene upstream of F.

[0165] In another emodiment, the present invention provides vaccinationor immunization against infectious bovine rhinotracheitis (IBR).

[0166] The virus responsible for infectious bovine rhinotrachitis is abovine herpesvirus type 1 (BHV-1), a member of the Alphaherpesvirinaefamily (Babiuk L. A. et al., 1996, Vet. Microbiol., 53, 31-42).Nucleotide sequences encoding the glycoproteins gB, gC and gD are knownand are accessible from the GenBank database under the accession numberAJ004801. The DNA vaccine or immunogenic or immunological compositionagainst BHV-1 may thus comprise DNA plasmid or plasmids comprising thenucleotide sequence(s) encoding the gB, gC and/or gD proteins.

[0167] The DNA vaccine or immunogenic or immunological compositionagainst BHV-1 as described above advantageously contains a veterinarilyacceptable vehicle or excipient according to the above-discussion of theinvention.

[0168] The DNA vaccine or immunogenic or immunological compositionagainst BHV-1 as described above can comprises an adjuvant according tothe above-discussion of the invention, such as DMRIE, advantageouslyDMRIE-DOPE.

[0169] Optionally, these embodiments may optionally further include (1)the addition of GM-CSF or of an expression vector, advantageously aplasmid, expressing GM-CSF, or (2) the optimization of at least oneBHV-1 antigen, or (3) the addition of bovine GM-CSF or of an expressionvector, advantageously a plasmid, expressing GM-CSF and the optimizationof at least one BHV-1 antigen. GM-CSF is advantageously bovine GM-CSF.The addition of GM-CSF may be carried out as described for BRSV and inthe above general discussion of the invention.

[0170] The optimization of the antigens derived from BHV-1 is carriedout by substitution, of a “signal” sequence of a BHV-1 antigen with aheterologous “signal” sequence, e.g., the human tPA signal sequence(GenBank accession number NM_(—)000930); for instance, substitution ofthe sequence of the signal peptide of the glycoprotein gB and/or of theglycoprotein gC and/or of the glycoprotein gD with the human tPA signalsequence; and/or by the deletion of the DNA fragment encoding thetransmembrane domain of gB and/or of gC and/or of gD. The deletion ofthe DNA fragment encoding the transmembrane domain of one of theseglycoproteins is advantageously accompanied by deletion of thecytoplasmic tail. The DNA vaccine or immunogenic or immunologicalcomposition against BHV-1 according to the invention can thereforeencode and express a single optimized BHV-1 antigen (gB, gC or gD) ortwo of them or all three, e.g., optimized gB, optimized gC and optimizedgD.

[0171] Nucleotide sequences encoding the BHV-1 antigens which can beused in the present invention and various constructs of expressionvectors are given herein, e.g. the accompanying examples, and inFR-A1-2751229, such as in Examples 7 and 8, and in FIGS. 3 and 4 (seealso U.S. Pat. No. 6,376,473 and U.S. application Ser. No. 10/085,519).

[0172] Advantageously, according to the invention, the DNA vaccine orimmunogenic or immunological composition against BHV-1 is formulatedwith DMRIE-DOPE, and is composed of an expression plasmid (e.g. pPB28 1,Example 3.1.2) encoding the BHV-1 gB antigen optimized by the deletionof the fragment of the nucleotide sequence encoding the transmembranedomain and the cytoplasmic tail, of a second expression plasmid (e.g.pPB292, Example 3.2.2) encoding the BHV-1 gC antigen optimized by thedeletion of the fragment of the nucleotide sequence encoding thetransmembrane domain and the cytoplasmic tail, and of a third expressionplasmid (e.g. pPB284, Example 3.3.2) encoding the BHV-1 gD antigenoptimized by the deletion of the fragment of the nucleotide sequenceencoding the transmembrane domain and the cytoplasmic tail.

[0173] In yet another embodiment the present invention providesvaccination or immunization against the BVDV.

[0174] The BVDV virus is a pestivirus of the Flaviviridae family. It isuniversally distributed in bovine populations and manifests itself byfetal malformations, abortions or clinical respiratory (mucosal disease)and enteric (bovine viral diarrhea) symptoms.

[0175] The BVDV viruses are distinguishable by the seriousness of theclinical signs and two groups have been formed, the BVDVs type 1(inapparent or mild clinical signs) and those of type 2 (acute clinicalsigns, hemorrhage, high morbidity, high mortality) (Dean H.J. and LeyhR., 1999, Vaccine, 17, 1117-1124).

[0176] When a BVDV type is not clearly specified, this virus isunderstood to be type 1 or type 2.

[0177] The BVDV is an enveloped single-stranded RNA virus composed of asingle gene encoding a polyprotein which, after cleavage, gives severalwell-individualized proteins, e.g., the E0 protein (gp48) and the E2protein (gp53) (Vassilev V. B. et al., 1997, J. Virol., 71, 471-478).

[0178] Nucleotide sequences encoding the E0-E2 polyproteins are knownand accessible from the GenBank database under the accession numberM96687 for BVDV-1 and AF145967 for BVDV-2. The DNA vaccine orimmunogenic or immunological composition against BVDV may thus compriseDNA plasmid or plasmids containing nucleotide sequence(s) encoding theE0 and/or E2 proteins.

[0179] The DNA vaccine or immunogenic or immunological compositionagainst BVDV as described above advantageously contain a veterinarilyacceptable vehicle or excipient according to the above-discussion of theinvention.

[0180] The DNA vaccine or immunogenic or immunological compositionagainst BVDV as described above can comprise an adjuvant according tothe above-discussion of the invention, such DMRIE, advantageouslyDMRIE-DOPE.

[0181] These two embodiments may further include (1) the addition ofbovine GM-CSF or of an expression vector, advantageously plasmidexpressing GM-CSF, or (2) the optimization of at least one BVDV antigen,or (3) the addition of bovine GM-CSF or of an expression vector,advantageously plasmid expressing GM-CSF and the optimization of atleast one BVDV antigen. GM-CSF is advantageously bovine GM-CSF. Theaddition of GM-CSF may be carried out as is described for BRSV and asdescribed in the above general discussion of the invention.

[0182] The optimization of the antigens derived from BVDV is carried outby the addition of a “signal” sequence, such as a heterologous signalsequence, advantageously the human tPA signal sequence, advantageouslyupstream of the nucleotide sequence encoding the E0 protein of BVDVand/or the E2 protein of BVDV, and/or by the deletion of the DNAfragment encoding the transmembrane domain of E2, and/or by theinsertion of an intron, such as intron II of the rabbit beta-globin,advantageously upstream of the nucleotide sequence encoding E0 and/orE2. The DNA vaccine or immunogenic or immunological composition againstBVDV according to the invention may therefore encode and express asingle optimized BVDV antigen (E0 or E2) or both (E0 and E2).

[0183] Nucleotide sequences encoding the BVDV antigens which can be usedin the present invention and various constructs of expression vectorsare given herein, e.g. the accompanying examples, and in FR-A1-2751229,such as in Example 13, and in FIG. 9 (see also U.S. Pat. No. 6,376,473and U.S. application Ser. No. 10/085,519).

[0184] Advantageously, according to the invention, the DNA vaccine orimmunogenic or immunological composition against BVDV is formulated withDMRIE-DOPE, and is composed of an expression plasmid (e.g. pLF1029Example 5.1.2, pLF1031 Example 6.2.2) encoding the E0 antigen of BVDVoptimized by the insertion of the signal sequence of the human tPAupstream of E0 and by the insertion of intron II of the rabbitbeta-globin gene upstream of E0, and of a second expression plasmid(e.g. pLF 1021 Example 5.2.2, pLF1023 Example 6.1.2) encoding the E2antigen of BVDV optimized by the insertion of the signal sequence of thehuman tPA upstream of E2, by the deletion of the fragment of thenucleotide sequence encoding the transmembrane domain of E2 and thecytoplasmic tail and by the insertion of intron II of the rabbitbeta-globin gene upstream of E2.

[0185] A mixture of plasmids can be advantageously produced and employedin the practice of the invention, e.g., the DNA vaccine or immunogenicor immunological composition can comprise a mixture of plasmids. Themixture may comprise at least two expression plasmids, each expressing adifferent immunogen (E0 or E2) and/or obtained from a different type ofBVDV (BVDV-1 or BVDV-2). A mixture can comprise four plasmids:oneexpressing BVDV-1 E0, one expressing BVDV-1 E2, one expressing BVDV-2 E0and one expressing BVDV-2 E2.

[0186] In an embodiment, the boost is done with an inactivated orattenuated or subunit vaccine or immunogenic or immunologicalcomposition. Inactivated or attenuated or subunit vaccines are availableto the person skilled in the art (e.g. see Ellis et al., J. Am. Vet.Med. Assoc., 2001, 218(12), 1973-1980 for BRSV; GB Patent No. 1,131,851for bPI-3; U.S. Pat. No. 5,676,951 and Published U.S. Application No.2002/0187929 for BHV; U.S. Pat. No. 6,291,228 for BVDV). In addition onemay use any commercial inactivated or attenuated or subunit vaccines,like BAR VAC® RS (Boehringer) for BRSV, VIROBOV H® (Merial) for bPI-3,IFFAVAX® I.B.R. (Merial) for BHV, BOVILIS BVD® (Intervet) or MUCOBOVIN®(Merial) for BVDV.

[0187] Advantageously, the inactivated or attenuated or subunit vaccineor immunogenic or immunological composition comprises an adjuvant, e.g.,an adjuvant as herein discussed.

[0188] In another embodiment, the boost is done with a recombinantvaccine or immunogenic or immunological composition which comprises anin vivo expression vector, such as a poxvirus, an adenovirus or aherpesvirus.

[0189] The expression vector comprises and expresses a nucleotidesequence encoding an immunogen from a bovine pathogen such as BRSV,bPI-3, BHV-1 and BVDV. The vector comprises and expresses at least oneimmunogen in common with the DNA vaccine or immunogenic or immunologicalcomposition. For these immunogens and the corresponding nucleotidesequences coding for these immunogens, reference is made to the hereindescription in relation with DNA vaccine or immunogenic or immunologicalcomposition. The nucleotide sequences may also be modified and improvedas described herein.

[0190] Specific, non-limiting examples include recombinant poxvirus,including avipox viruses, such as canarypox virus (U.S. Pat. No.5,505,941) and vaccinia viruses (U.S. Pat. No. 4,603,112), such asattenuated vaccinia virus such as NYVAC (see U.S. Pat. No. 5,494,807) orModified Vaccinia virus Ankara (MVA, Stickl H. and Hochstein-Mintzel V.,Munch. Med. Wschr. 113:1149-1153, 1971; Sutter G. et al., Proc. Natl.Acad. Sci. U.S.A. 89:10847-10851, 1992; Carroll M. W. et al., Vaccine15(4):387-394, 1997; Stittelaar K. J. et al., J. Virol. 74(9):4236-4243,2000; Sutter G. et al., Vaccine 12(11):1032-1040, 1994). When avipoxviruses are used, dovepox viruses, canarypox viruses (U.S. Pat. No.5,756,103) and fowlpox viruses (U.S. Pat. No. 5,766,599) may beemployed, such as attenuated viruses canarypox viruses and attenuatedfowlpox viruses, for instance ALVAC and TROVAC. For recombinantcanarypox virus vectors, the insertion sites may be the ORFs C3, C5 orC6. When the expression vector is a poxvirus, the heterologouspolynucleotide can be inserted under the control of a poxvirus specificpromoter, such as the vaccinia virus 7.5 kDa promoter (Cochran et al.,J. Virology 54:30-35, 1985), the vaccinia virus 13L promoter (Riviere etal., J. Virology 66:3424-3434, 1992), the vaccinia virus HA promoter(Shida, Virology 150:451-457, 1986), the cowpox virus ATI promoter(Funahashi et al., J. Gen. Virol. 69:35-47, 1988), the vaccinia virus H6promoter (Taylor et al., Vaccine 6:504-508, 1988; Guo et al., J. Virol.63:4189-4198, 1989; Perkus et al., J. Virol. 63:3829-3836, 1989).

[0191] Other useful viral vectors include herpesvirus or adenovirusvectors. Specific, non-limiting examples include bovine herpesvirus(BHV) or bovine adenovirus (BAV) as a vector (for example, see PublishedEuropean Application No. EP 0.663.403; Published PCT Application No. WO98/59063). For BHV, the insertion sites may be the thymidine kinasegene, in the gE, or in the gI (see Published PCT Application No. WO92/21751). For BAV, the insertion sites may be the E2 region or in theE4 region (see U.S. Pat. No. 6,451,319; U.S. Pat. No. 6,319,716). In BHVor BAV vectors the insert (heterologous nucleic acid molecule encodingthe immunogen, antigen or epitope of interest, e.g., of a bovinepathogen, such as that which is expressed by the DNA vaccine orimmunogenic composition) is, in general, under the control of (oroperably linked to) a promoter. The promoter may be of viral or cellularorigin. The cytomegalovirus (CMV) early promoter (CMV-IE promoter),including the promoter and enhancer, may used. The CMV-IE promoter canbe of human or murin origin, or optionally of other origin such as rator guinea pig (see EP 0260148; EP 0323597; WO 89/01036; Pasleau et al.,Gene 38:227-232, 1985; Boshart M. et al., Cell 41:521-530, 1985); seealso discussion above (concerning promoters for use in DNA plasmids).Functional fragments of the CMV-IE promoter may also be used (WO98/00166); see also discussion above (concerning promoters for use inDNA plasmids). The SV40 virus early or late promoter and the RousSarcoma virus LTR promoter may also be used. Other promoters include butare not limited to, a promoter of the cytoskeleton gene, such as thedesmin promoter (Kwissa M. et al., Vaccine 18(22):2337-2344, 2000), orthe actin promoter (Miyazaki J. et al., Gene 79(2):269-277, 1989).Advantageously the promoter is a CMV-IE promoter.

[0192] The inactivated or attenuated or subunit vaccine or immunogenicor immunological composition, or the recombinant vaccine or immunogenicor immunological composition can be supplemented with an adjuvant suchas fMLP (N-formyl-methionyl-leucyl-phenylalanine; U.S. Pat. No.6,017,537) and/or acrylic acid or methacrylic acid polymer and/or acopolymer of maleic anhydride and of alkenyl derivative. The acrylicacid or methacrylic acid polymers can be cross-linked, e.g., withpolyalkenyl ethers of sugars or of polyalcohols. These compounds areknown under the term “carbomer” (Pharmeuropa, Vol. 8, No. 2, June 1996).A person skilled in the art may also refer to U.S. Pat. No. 2,909,462(incorporated by reference) which discusses such acrylic polymerscross-linked with a polyhydroxylated compound containing at least 3hydroxyl groups: in one embodiment, a polyhydroxylated compound containsnot more than 8 hydroxyl groups; in another embodiment, the hydrogenatoms of at least 3 hydroxyls are replaced with unsaturated aliphaticradicals containing at least 2 carbon atoms; in other embodiments,radicals contain from about 2 to about 4 carbon atoms, e.g., vinyls,allyls and other ethylenically unsaturated groups. The unsaturatedradicals can themselves contain other substituents, such as methyl. Theproducts sold under the name Carbopol® (Noveon Inc., Ohio, USA) areparticularly suitable for use as an adjuvant. They are cross-linked withan allyl sucrose or with allylpentaerythritol, as to which, mention ismade of the products Carbopol® 974P, 934P, and 971P.

[0193] As to the copolymers of maleic anhydride and of alkenylderivative, mention is made of the EMA® products (Monsanto) which arecopolymers of maleic anhydride and of ethylene, which may be linear orcross-linked, for example cross-linked with divinyl ether. Also,reference may be made to J. Fields et al., Nature 186:778-780, 1960(incorporated by reference). Generally, the acrylic acid or methacrylicacid polymers, such as the carbomers, and the copolymers of malcicanhydride and of alkenyl derivative, such as the EMA® products, areformed from units based on the following formula:

[0194] in which:

[0195] R₁ and R₂, which may be identical or different, represent H orCH₃

[0196] x=0 or 1, advantageously, x=1

[0197] y=1 or 2, with x+y=2.

[0198] For the EMA® products, x=0 and y=2. For the carbomers, x=y=1.

[0199] The dissolution of these polymers in water leads to an acidsolution, which is neutralized to physiological pH, in order to give theadjuvant solution into which the immunogenic composition or the vaccineitself is incorporated. The carboxyl groups of the polymer are thenpartly in COO⁻ form.

[0200] Advantageously, a solution of adjuvant, e.g., carbomer, isprepared in distilled water, for example, in the presence of a salt suchas sodium chloride; the solution obtained is at acidic pH. This stocksolution is diluted by adding it to the desired quantity (for obtainingthe desired final concentration), or a substantial part thereof, ofwater containing a salt such as NaCl, advantageously physiologicalsaline (NaCL 9 g/l) all at once or in several portions with concomitantor subsequent neutralization (pH 7.3 to 7.4). The stock solution isneutralized with a base such as NaOH. This solution of adjuvant atphysiological pH is used as it is for mixing with the immunogeniccomposition or with the vaccine, which may be especially stored infreeze-dried, liquid or frozen form.

[0201] The polymer concentration in the final vaccine composition can befrom about 0.01% to about 1.5% W/V. The final vaccine composition can befrom about 0.05 to about 1% W/V. The final vaccine composition can befrom about 0.1 to about 0.4% W/V.

[0202] The inactivated or attenuated or subunit or recombinant vaccineor immunogenic or immunological composition can also be formulated inthe form of an oil-in-water emulsion. The oil-in-water emulsion can bebased, for example, on light liquid paraffin oil (European Pharmacopeatype); isoprenoid oil such as squalane, squalene, EICOSANE™ ortetratetracontane; oil resulting from the oligomerization of alkene(s),e.g., isobutene or decene; esters of acids or of alcohols containing alinear alkyl group, such as plant oils, ethyl oleate, propylene glycoldi(caprylate/caprate), glyceryl tri(caprylate/caprate) or propyleneglycol dioleate; esters of branched fatty acids or alcohols, e.g.,isostearic acid esters. The oil advantageously is used in combinationwith emulsifiers to form the emulsion. The emulsifiers can be nonionicsurfactants, such as esters of sorbitan, mannide (e.g., anhydromannitololeate), glycerol, polyglycerol, propylene glycol, and oleic,isostearic, ricinoleic, or hydroxystearic acid, which are optionallyethoxylated, and polyoxypropylene-polyoxyethylene copolymer blocks, suchas the Pluronic® products, e.g., L121. In one specific, non-limitingexample, the oil is provided in an amount between about 1 and about 60%.The oil can be in an amount between about 5 and about 30%. The adjuvantcan be a mixture of emulsifier(s), micelle-forming agent, and oil suchas that which is available under the name Provax® (IDEC Pharmaceuticals,San Diego, Calif.).

[0203] The DNA plasmid, the inactivated or attenuated or subunit or theexpression, e.g., viral, vector according to this disclosure can bepreserved and/or conserved and stored either in liquid form, e.g., atlow temperature, e.g. at about 5° C., or in lyophilised or freeze-driedform, e.g., in the presence of a stabilizer. Freeze-drying can beaccording to well-known standard freeze-drying procedures. Thepharmaceutically acceptable stabilizers may be SPGA (sucrose phosphateglutamate albumin) (Bovarnik et al., J. Bacteriology 59:509, 1950),carbohydrates (e.g., sorbitol, mannitol, lactose, sucrose, glucose,dextran, trehalose), sodium glutamate (Tsvetkov T et al., Cryobiology20(3):318-23, 1983; Israeli E et al., Cryobiology 30(5):519-23, 1993),proteins such as peptone, albumin or casein, protein containing agentssuch as skimmed milk (Mills C K et al., Cryobiology 25(2):148-52, 1988;Wolff E et al., Cryobiology 27(5):569-75, 1990), and buffers (e.g.,phosphate buffer, alkaline metal phosphate buffer). An adjuvant and/or avehicle or excipient may be used to make soluble the freeze-driedpreparations.

[0204] Another aspect of the present invention is the use of plasmidscontaining and expressing in vivo in a bovine at least one immunogenfrom a bovine pathogen, e.g., BRSV, bPI-3, BHV-1 or BVDV, for thepreparation of a DNA vaccine, e.g., for use in a prime-boost method ofthe invention and/or for a kit for a prime-boost method of the inventionand/or to induce an immune response in a young bovine, e.g., calve,which have or may have maternal antibodies against the bovine pathogen.

[0205] Advantageously, the DNA vaccine is to be administered (and isadministered) to the young animal (bovine) from calving up to andincluding about 12 weeks of age, such as from calving up to andincluding 6 weeks of age, for instance, from calving up to and including4 weeks of age, e.g., from calving up to and including 3 weeks of age.

[0206] More advantageously, the DNA vaccine is intended to induce (andinduces) a priming immune response specific for the expressed immunogenor a “DNA induced immune response” (such as agamma-interferon+(IFN_(γ)+) memory T cell response specific for theexpressed immunogen), which is boostable (can be boosted), by asubsequent administration (boost) of an inactivated or attenuated orsubunit vaccine or a recombinant or modified vector (e.g., viral,bacterial) vaccine or immunogenic composition comprising a vector, e.g.,viral vector, such as a live recombinant poxvirus, containing andexpressing in vivo at least the same immunogen(s), antigen(s) orepitope(s) as that expressed by the DNA vaccine.

[0207] The boost administration may be administered from about 2 weeksto about 5 months after the priming administration, such as from about 3to about 6 weeks after the priming administration, and advantageouslyabout 4 weeks after the priming administratoin. A second administrationof the boost vaccine or immunological or immunogenic composition mayoccur, for instance, when the calve is transferred to a finishing unit.

[0208] In another aspect, the present invention involves the use of abovine pathogenic agent (including a fragment thereof), such as BRSV,bPI-3, BHV-1 or BVDV, for the preparation of a priming vaccine based onplasmids (DNA vaccine or immunogenic or immunological composition)containing and expressing in. vivo in a bovine (e.g., cow, bull, calve,cattle) a nucleic acid molecule encoding at least one immunogen, antigenor epitope from the pathogenic agent, and for the preparation of asecond vaccine (a boost vaccine or immunogenic or immunologicalcomposition) comprising the pathogenic agent in an inactivated form, orin an attenuated form, or wherein the second vaccine comprises a subunit(isolated protein, antigen, immunogen or epitope) of the pathogenicagent or wherein the second vaccine comprises a recombinant or modifiedexpression vector (e.g., viral or bacterial or yeast vector), such as alive recombinant poxvirus, advenovirus, or herpesvirus, advantageously apoxvirus, that contain(s) and express(es) in vivo nucleic acidmolecule(s) encoding at least the immunogen(s), antigen(s) or epitope(s)of the pathogenic agent, including such immunogen(s), antigen(s) orepitope(s) expressed by the plasmid-based vaccine. Here, “use of apathogenic agent” encompasses the use of the pathogen to clone thenucleotide sequence encoding the immunogen, antigen or epitope, as wellas the use of the pathogenic agent to isolate therefrom the immunogen,antigen or epitope (e.g., for the subunit or to sequence the same toascertain a coding sequence therefore for preparation of the DNA plasmidor the recombinant or modified expression vector), as well as the use ofthe pathogenic agent to prepare the inactivated or attenuated vaccine orimmunogenic composition. Thus, another aspect of the present inventionis the use of a nucleotide sequence coding for at least one immunogen,antigen or epitope of a bovine pathogenic agent, such as BRSV, bPI-3,BHV-1 or BVDV, for the preparation of the priming vaccine based onplasmid (DNA vaccine or immunogenic or immunological composition) and/orthe second vaccine (or immunogenic or immunological composition) thatcontains the modified or recombinant expression vector. The subunit,inactivated or attenuated vaccine or immunogenic compositionadvantageously contains the immunogen, antigen or epitope expressed bythe DNA vaccine or immunogenic or immunological composition; and, therecombinant or modified expression vector advantageously expresses theimmunogen, antigen or epitope expressed by the DNA vaccine orimmunogenic or immunological composition. The plasmid-based vaccine isintended to be administered to a bovine first (such as to a young calvewhich has or may have maternal antibodies against the bovine pathogen),and the inactivated or attenuated or subunit or recombinant or modifiedexpression vector vaccine is intended to be administered after the DNAvaccine and to the same bovine, to boost the immune response against theimmunogen, antigen or epitope (for instance, at the time the inactivatedor attenuated or subunit or recombinant or modified expression vectorvaccine is administered, the calve has developed a specific primingimmune response against the immunogen(s), antigen(s) or epitope(s), suchas a specific DNA vaccine immune response or “DNA vaccine induced”immune response against the immunogen(s), antigen(s), or epitope(s),e.g., the IFN_(γ)+ memory T cell response specific for the expressedimmunogen, antigen or epitope; and the inactivated or attenuated orsubunit or recombinant or modified expression vector vaccine induces animmune response against the bovine pathogen including against at leastone of the the immunogen(s), antigen(s) or epitope(s) expressed by theDNA vaccine).

[0209] Accordingly another aspect of the present invention is the use ofa bovine pathogenic agent, such as BRSV, bPI-3, BHV-1 or BVDV, toprepare an inactivated or attenuated or subunit vaccine or immunogeniccomposition to vaccinate or immunize a bovine against the pathogenicagent, wherein the bovine (e.g., a young calve which has or may havematernal antibodies against the bovine pathogen) has previously beenimmunized with a DNA vaccine that expresses in vivo at least oneimmunogen, antigen or epitope from the same pathogenic agent and hasdeveloped a specific priming immune response against the immunogen(s),antigen(s) or epitope(s), such as the “DNA vaccine induced” immuneresponse, more advantageously the IFN_(γ)+ memory T cell responsespecific for the expressed immunogen, antigen or epitope.

[0210] Likewise, another aspect of the present invention is the use of arecombinant or modified expression vector, e.g., a viral vector, such asa poxvirus vector, that comprise(s) and express(es) in vivo at least onenucleotide sequence coding for at least one immunogen, antigen orepitope from a bovine pathogenic agent, such BRSV, bPI-3, BHV-1 or BVDV,to prepare a recombinant or modified expression vector (e.g., a liverecombinant or modified vector) vaccine or immunogenic composition tovaccinate a bovine against the pathogenic agent, wherein the bovine(such as a young calve which has or may have maternal antibodies againstthe bovine pathogen) has previously been immunized with a DNA vaccinethat expresses in vivo at least the same immunogen(s), antigen(s) orepitope(s) and has developed a priming immune response against theimmunogen(s), antigen(s) or epitope(s), such as the “DNA vaccineinduced” immune response, more advantageously, the IFN_(γ)+ memory Tcell response specific for the expressed immunogen, antigen or epitope.

[0211] The DNA vaccine is advantageously administered to the younganimal (bovine) from calving up to and including about 12 weeks of age,such as from calving up to and including about 6 weeks of age, forinstance, from calving up to and including 4 weeks of age, e.g., fromcalving up to and including 3 weeks of age. The inactivated orattenuated or subunit or recombinant or modified expression vectorvaccine or immunogenic composition is intended to be administered fromabout 2 weeks to about 5 months after the priming administration, suchas from about 3 to 6 weeks after, and advantageously about 4 weeksafter. A second administration of the boost vaccine or immunological orimmunogenic composition may occur, such as when the calve is transferredto a finishing unit.

[0212] The inactivated or the recombinant or modified expression vectorvaccine or immunogenic composition is advantageously employed in thepractice of the invention. The inactivated or attenuated or subunit orrecombinant or modified expression vector vaccine or immunogeniccomposition can be as described herein and can advantageously comprisean adjuvant. To avoid repitition, it is generally mentioned thatdiscussion elsewhere in this description may be applied to the herein“use” aspects of the invention.

[0213] The method and uses of the invention may combine immunization orvaccination against more than one bovine pathogen, and the methods anduses of the invention may encompass any combination immunization orvaccination against 2, 3 or 4 of the particular herein mentionedpathogens. This may be performed by concomitant or successiveadministration of the vaccines or immunogenic or immunologic compositionagainst the pathogens. This may also involve mixtures of thecorresponding immunogens or vaccines or immunogenic or immunologiccomposition. This may also involve plasmids or vectors comprising andexpressing nucleic acid molecules encoding immunogens, antigens orepitopes of two or more or several pathogens. Accordingly, anotheraspect of the invention is multivalent DNA vaccines or immunogenic orimmunological compositions.

[0214] Still another aspect of the invention is a kit containing a firstvaccine or immunogenic or immunological composition which comprises aDNA vaccine or immunogenic or immunological composition according to theinvention and a second vaccine or immunogenic or immunologicalcomposition comprising an inactivated, attenuated live, subunit,advantageously inactivated vaccine or immunogenic or immunologicalcomposition, or recombinant or modified in vivo expression vectorvaccine or immunogenic or immunological composition, wherein the subunitcontains an immunogen or antigen or epitope expressed by the firstvaccine and the modified vector vaccine or immunogenic or immunologicalcomposition expresses an immunogen, antigen or epitope expressed by thefirst vaccine, and the attenuated vaccine or immunogenic orimmunological composition expresses or presents an immunogen, antigen orepitope expressed by the first vaccine, and the inactivated vaccine orimmunogenic or immunological composition presents an immunogen, antigenor epitope expressed by the first vaccine. The vaccine(s) orcomposition(s) are advantageously in separate containers. The separatecontainers can be packaged together. The kit can contain instructionsfor prime-boost administration according to the invention.

[0215] The quantity of DNA used in the vaccines and compositionsaccording to the present invention is advantageously between about 1 μgand about 1000 μg, and such as between about 50 μg and about 500 μg, fora given plasmid. Persons skilled in the art possess the competencenecessary to precisely define the effective dose of DNA to be used foreach vaccination protocol, from this disclosure and the knowledge in theart.

[0216] The dose volumes may be between about 0.2 and about 5 ml,advantageously between about 1 and about 3 ml.

[0217] The DNA vaccines and compositions according to the invention maybe administered, in the context of th vaccination method of theinvention, by various routes of administration proposed in the art forpolynucleotide vaccination and by means of known techniques ofadministration.

[0218] According to a mode of the invention, the DNA vaccines orcompositions according to the invention are administered by theintramuscular route, the subcutaneous route or with the aid of aneedleless injector such as the Biojector 2000 (Bioject Inc., PortlandOreg., USA), advantageously by the intradermal route. For more detailson administration to a bovine via the intradermal route using aneedleless injector, one may refer to U.S. Pat. No. 6,451,770.

[0219] For the boost administration with a recombinant or modified invivo expression vector vaccine or immunogenic or immunologicalcomposition (e.g., a recombinant virus such as a recombinantherpesvirus, adenovirus, or poxvirus composition, advantageously arecombinant poxvirus composition, such as a recombinant vaccinia,avipox, canarypox fowlpox virus composition, advantageously an ALVAC,TROVAC, or NYCAC composition), the route of administration can beintradermal (ID), intramuscular (IM), subcutaneous (SC), intravenous,oral or nasal. This administration can be made with a syringe and aneedle or with a needleless injector. The dosage is advantageously fromabout 10³ pfu to about 10⁹ pfu per recombinant vector. When the vectoris a canarypox virus, the dosage is advantageously from about 10⁵ pfu toabout 10⁹ pfu, e.g. from about 10⁶ pfu to about 10⁷ pfu. The volume ofneedleless injector doses could be between about 0.1 ml and about 0.5ml, e.g. about 0.25 ml. For injection with a syringe and a needle, thevolumes advantageously can be from about 0.5 to about 5 ml, e.g. about 1to about 3 ml. The dosage can be as mentioned herein.

[0220] For the boost administration with an inactivated, attenuated orsubunit vaccine or immunogenic or immunological compositions, the routeof administration can be intradermal (ID), intramuscular (IM),subcutaneous (SC), intravenous, oral or nasal. This administration canbe made with a syringe and a needle or with a needleless injector. Thevolume of needleless injector doses advantageously can be between about0.1 ml and about 0.5 ml, e.g. about 0.25 ml. For injection with asyringe and a needle, the volumes advantageously can be from about 0.5to about 5 ml, e.g. from about 1 to about 3 ml.

[0221] The invention will now be further described and illustrated byway of the following, non-limiting examples.

EXAMPLES

[0222] For each of the pathogens considered, each gene encoding theprincipal antigens (native form and modified form) was the subject of aparticular construction in a eukaryotic expression plasmid. The secretedforms of the antigens were obtained by deletion of the fragments ofgenes encoding the transmembrane and cytoplasmic domains. In all cases,the transmembrane domains of the proteins were identified on the basisof the hydropathy profiles (on MacVector 6.5) of the correspondingprotein sequences.

Example 1 Molecular biology methods

[0223] 1.1 Extraction of Viral Genomic DNA

[0224] Viral suspensions were treated with proteinase K (100 mg/mlfinal) in the presence of sodium dodecyl sulphate (SDS) (0.5% final) for2 hours at 37° C. The viral DNA was then extracted with the aid of aphenol/chloroform mixture, and then precipitated with two volumes ofabsolute ethanol at −20° C. for 16 hours and then centrifuged at 10,000g for 15 minutes at 4° C. The DNA pellets were dried, and then taken upin a minimum volume of sterile ultrapure water.

[0225] 1.2 Isolation of Viral Genomic RNA

[0226] The genomic RNA of each virus was extracted using the“guanidinium thiocyanate/phenol-chloroform” technique described by P.Chomczynski and N. Sacchi (Anal. Biochem. 1987. 162. 156-159).

[0227] 1.3 Molecular Biology Techniques

[0228] All the constructions of plasmids were carried out using thestandard molecular biology techniques described by Sambrook et al.(Molecular Cloning: A Laboratory Manual. 2nd Edition. Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y., 1989). All the restrictionfragments used for the present invention were isolated with the aid ofthe “Geneclean” kit (BIO101 Inc., La Jolla, Calif.). For all theconstructs, the cloned DNA fragments, as well as the junctions with theexpression vector, were sequenced by the Sanger method (Sambrook et al.,1989).

[0229] 1.4 PCR and RT-PCR

[0230] The oligonucleotides specific to the genes or gene fragmentscloned were synthesized, some of them containing, in some cases, attheir 5′ end, restriction sites facilitating the cloning of theamplified fragments. The reverse transcription (RT) reactions and thepolymerase chain reaction (PCR) were carried out according to standardtechniques (Sambrook et al., 1989).

[0231] 1.5 Large-Scale Purification of Plasmids

[0232] The production, on the scale of about ten mg, of purifiedplasmids entering into the vaccinal compositions was carried out by thecaesium chloride-ethidium bromide gradient method (Sambrook et al.,1989).

Example 2 Basic Plasmid Constructs

[0233] The eukaryotic expression plasmid pVR1020 (C. J. Luke et al. J.of Infectious Diseases, 1997, 175, 95-97), derived from the plasmidpVR1012 (FIG. No. 1, FIG. 1 and Example 7 of WO-A-9803 199), containsthe coding phase of the signal sequence of the human tissue plasminogenactivator (tPA).

[0234] A plasmid pVR1020 is modified by BamHI-BglII digestion andinsertion of a sequence containing several cloning sites (BamHI, NotI,EcoRI, XbaI, PmII, PstI, BglII) and resulting from the pairing of thefollowing oligonucleotides:

[0235] PB326 (40 mer) (SEQ ID NO 1)

[0236] 5′GATCTGCAGCACGTGTCTAGAGGATATCGAATTCGCGGCC 3′ and

[0237] PB329 (40 mer) (SEQ ID NO 2)

[0238] 5′GATCCGCGGCCGCGAATTCGATATCCTCTAGACACGTGCT 3′.

[0239] The vector thus obtained, having a size of about 5105 base pairs(or bp), is called pAB110 (FIG. No. 2).

[0240] Intron II of the rabbit β-globin gene is cloned into the vectorpCRII (Invitrogen, Carlsbad, Calif., USA) after production of thecorresponding DNA fragment by PCR with the aid of the followingoligonucleotides:

[0241] SB090 (20 mer) (SEQ ID NO 3)

[0242] 5′TTGGGGACCCTTGATTGTTC 3′ and

[0243] SB091 (21 mer) (SEQ ID NO 4)

[0244] 5′CTGTAGGAAAAAGAAGAAGGC 3′

[0245] using as template the genomic DNA of rabbit peripheral bloodcells. The resulting plasmid is designated pNS050.

[0246] The expression plasmid pAB110 is modified by introducing thesequence of intron II of the rabbit globin gene into the SalI sitesituated upstream of the ATG of the signal peptide of tissue plasminogenactivator (tPA). The sequence of intron II of the rabbit globin gene isamplified by polymerase chain reaction (PCR) from the plasmid pNS050using the following oligonucleotide pair:

[0247] LF001 (30 mer) (SEQ ID NO 5)

[0248] 5′CTCCATGTCGACTTGGGGACCCTTGATTGT 3′ and

[0249] LF002 (30 mer) (SEQ ID NO 6)

[0250] 5′CTCCATGTCGACCTGTAGGAAAAAGAAGAA 3′

[0251] The PCR product (573 base pairs or bp) is digested with SalI andcloned into the plasmid pAB110 previously linearized with SalI, togenerate the plasmid pLF999 of about 5678 bp.

Example 3 Plasmids Encoding the Various Forms of the Bovine HerpesvirusType 1 (BHV-1) Antigens

[0252] Fragments of viral DNA containing the gB, gC and gD genes of theB901 strain of BHV-1 are isolated by digesting the viral genome withvarious restriction enzymes, by separating them by agarose gelelectrophoresis and by analysing them by Southern blotting with the aidof probes corresponding to fragments of the gB, gC and gD genes of theST strain of BHV-1 (Leung-Tack P. et al., Virology, 1994, 199, 409-421).The BHV-1 Colorado strain [Cooper] (ATCC number VR-864) can also beused. The fragments thus identified are cloned into the vectorpBluescript SK+ (Stratagene, La Jolla, Calif., USA) and are at theorigin of the clonings of the three genes into the expression vectorpVR1012.

[0253] 3.1 Plasmids Encoding the Various Forms of BHV-1 gB

[0254] 3.1.1 pPB280: gB Gene (Native Form) Cloned Into the VectorpVR1012

[0255] Two XhoI-XhoI fragments containing the 5′ and 3′ portions of theBHV-1 gB gene are identified by Southern blotting and cloned into thevector pBluescript SK+ (Stratagene, La Jolla, Calif., USA) previouslydigested with XhoI. The plasmids thus obtained are designated pPB128 andpPB117 respectively.

[0256] The plasmid pPB128, containing the 5′ fragment of the gB gene, isdigested with NotI and XhoI, generating a fragment of 1708 bp (fragmentA).

[0257] The plasmid pPB117, containing the 3′ portion of the gB gene, isdigested with XhoI and StuI, generating a fragment of 1345 bp. Thelatter fragment is cloned into the vector pBluescript KS+ (Stratagene,La Jolla, Calif., USA) previously digested with EcoRV and XhoI. Theresulting plasmid is called pPB279. The plasmid pPB279 is then digestedwith XhoI and BamHI, generating a DNA fragment of 1413 bp (fragment B).

[0258] Fragments A and B are then cloned into a vector pBluescript KS+digested with NotI and BamHI, generating plasmid pPB278 (about 6063 bp)and allowing the reconstitution of the BHV-1 gB gene.

[0259] The vector pPB278 then serves as template during a PCR reactioncarried out with the following oligonucleotides:

[0260] PB234 (30 mer) (SEQ ID NO 7)

[0261] 5′ TTGTCGACATGGCCGCTCGCGGCGGTGCTG 3′ and

[0262] PB235 (21 mer) (SEQ ID NO 8)

[0263] 5′ GCAGGGCAGCGGCTAGCGCGG 3′.

[0264] The PCR product (146 bp) is then digested with the restrictionenzymes SalI and NheI.

[0265] The plasmid pPB278 is digested with NheI and BamHI. The fragmentof 2728 bp thus obtained and the PCR fragment previously digested areligated into the vector pVR1012 (Example 2) previously digested withSalI and BamHI, thus generating the plasmid pPB280, having a size ofabout 7742 bp.

[0266] The BHV-1 gB gene encodes a protein of 933 amino acids.

[0267] 3.1.2 pPB281: gB Gene (Δ[TM−Cter] form) cloned into the vectorpVR1012 The truncated form (deleted for its transmembrane (TM) andcarboxy-terminal (Cter) domains) of the BHV-1 gB gene is obtained byligating into the plasmid pVR1012 (Example 2) predigested with SalI andBamHI, both a fragment having a size of 2234 bp obtained after digestionwith SalI-PvuII of the plasmid pPB280 (Example 3.1.1) and a fragment of56 bp obtained by pairing of the following oligonucleotides:

[0268] PB511 (52 mer) (SEQ ID NO 9)

[0269] 5′CTGCACGAGCTCCGGTTCTACGACATTGACCGCGTGGTCAAGACGGACTGAG 3′ and

[0270] PB512 (57 mer) (SEQ ID NO 10)

[0271] 5′GATCCTCAGTCCGTCTTGACCACGCGGTCAATGTCGTAGAACCGGAGCTCGTGCA G3′.

[0272] The plasmid thus generated has a size of about 7154 bp and iscalled pPB281. The truncated gB gene of BHV-1 encodes a protein of 759amino acids.

[0273] 3.1.3 pSB115: gB Gene (tPA Δ[TM−Cter] form) cloned into thevector pAB110

[0274] The tPA Δ[TM−Cter] form of the BHV-1 gB gene is amplified by PCRfrom the template pPB281 (Example 3.1.2) and with the aid of thefollowing primers:

[0275] SB221 (39 mer) (SEQ ID NO 11)

[0276] 5′ AAAATTTCGATATCCGCCGCGGGGCGACCGGCGACAACG 3′ and

[0277] SB222 (33 mer) (SEQ ID NO 12)

[0278] 5′ GGAAGATCTTCAGTCCGTCTTGACCACGCGGTC 3′

[0279] The amplification product (2088 bp) is digested with the enzymesEcoRV and BglII and cloned into the vector pAB110 (Example 2) previouslydigested with EcoRV and BglII, generating the plasmid pSB115, having asize of about 7154 bp.

[0280] The tPA Δ[TM−Cter] form of the gB gene encodes a glycoprotein of729 amino acids, containing the extracellular domain of the BHV-1 gBglycoprotein.

[0281] 3.2. Plasmids Encoding the Various Forms of BHV-1 gC

[0282] 3.2.1 pPB264: gC Gene (Native Form) Cloned Into the VectorpVR1012

[0283] A BamHI-HindIII fragment of 3.5 kb containing the complete BHV-1gC gene is identifed by Southern blotting and cloned into the vectorpBluescript SK+. The plasmid thus obtained is called pPB287.

[0284] The plasmid pPB287 is then digested with NcoI-BssSI. A digestionfragment having a size of 1492 bp is obtained. It is ligated with asynthetic DNA fragment obtained by the pairing of the followingoligonucelotides:

[0285] PB507 (37 mer) (SEQ ID NO 13)

[0286] 5′ TCGTGCCTGCGGCGCAAGGCCCGGGCGCGCCTGTAGT 3′ and

[0287] PB508 (37 mer) (SEQ ID NO 14)

[0288] 5′ CTAGACTACAGGCGCGCCCGGGCCTTGCGCCGCAGGC 3′,

[0289] into the plasmid pLitmus 28 (New England Biolabs, Inc., Beverly,Mass., USA) predigested with NcoI and XbaI, generating the intermediateplasmid pPB290.

[0290] The fragment of 1554 bp derived from the digestion of pPB290 withPstI and XbaI is cloned into the vector pVR1012 (Example 2) previouslydigested with PstI and XbaI, thus generating the plasmid pPB264, havinga size of about 6427 bp. The BHV-1 gC gene encodes a protein of 508amino acids.

[0291] 3.2.2 pPB292: gC gene (Δ[TM−Cter] form) cloned into the vectorpVR1012

[0292] The truncated form of the BHV-1 gC gene is obtained by ligatingthe following three DNA fragments into the vector pVR1012 (Example 2)previously digested with PstI and XbaI:

[0293] (a) a fragment of 1035 bp derived from the digestion of pPB264(Example 3.2.1) with PstI and XhoI,

[0294] (b) a fragment of 350 bp derived from the digestion of pPB264with XhoI and BanI and

[0295] (c) a synthetic fragment of 43 bp resulting from the pairing ofthe oligonucleotides PB513 and PB514.

[0296] These oligonucleotides are the following:

[0297] PB513 (43 mer) (SEQ ID NO 15)

[0298] 5′ GCACCGCTGCCCGAGTTCTCCGCGACCGCCACGTACGACTAGT 3′ and

[0299] PB514 (43 mer) (SEQ ID NO 16)

[0300] 5′ CTAGACTAGTCGTACGTGGCGGTCGCGGAGAACTCGGGCAGCG 3′.

[0301] The plasmid having a size of about 6305 bp thus obtained iscalled pPB292. The truncated gC gene of BHV-1 encodes a protein of 466amino acids.

[0302] 3.2.3 pSB116: gC gene (tPA Δ[TM−Cter] form) cloned into thevector pAB110

[0303] The tPA Δ[TM−Cter] form of the BHV-1 gC gene is amplified by PCRfrom the template pPB292 (Example 3.2.2) and with the aid of thefollowing primers:

[0304] SB223 (39 mer) (SEQ ID NO 17)

[0305] 5′ AAAATTTCGATATCCCGGCGGGGGCTCGCCGAGGAGGCG 3′ and

[0306] SB224 (32 mer) (SEQ ID NO 18)

[0307] 5′ GGAAGATCTCTAGTCGTACGTGGCGGTCGCGG 3′

[0308] The amplification product (1362 bp) is digested with the enzymesEcoRV and BglII and cloned into the vector pAB110 (Example 2) previouslydigested with EcoRV and BglII, generating the plasmid pSB116, having asize of about 6404 bp.

[0309] The tPA Δ[TM−Cter] form of the gC gene encodes a glycoprotein of479 amino acids, containing the extracellular domain of the BHV-1 gCglycoprotein.

[0310] 3.3 Plasmids Encoding the Various Forms of BHV-1 gD

[0311] 3.3.1 pPB148: gD Gene (Native Form) Cloned Into the VectorpVR1012

[0312] A XhoI-XhoI fragment of 5 kb containing the BHV-1 gD gene isidentified by Southern blotting and cloned into the vector pBluescriptSK+predigested with XhoI, generating the plasmid pPB147.

[0313] A fragment of 325 bp derived from the digestion of pPB147 withNdeI and BsrBI and a fragment of 943 bp derived from the digestion ofpPB147 with NdeI and StyI are then ligated into the vector pVR112(Example 2) predigested with EcoRV and XbaI, thus generating the plasmidpPB 148, having a size of about 6171 bp. The BHV-1 gD gene encodes aprotein of 417 amino acids.

[0314] 3.3.2 pPB284: gD gene (Δ[TM−Cter] form) cloned into the vectorpVR1012 The truncated gD gene of BHV-1 is obtained from a fragmentobtained after PCR amplification carried out on the genomic DNA of theB901 strain of the BHV-1 virus previously digested with PstI and XbaIand with the aid of the following primer pair: PB497 (33 mer) (SEQ ID NO19)

[0315] 5′ TTTCTGCAGATGCAAGGGCCGACATTGGCCGTG 3′ and

[0316] PB498 (31 mer) (SEQ ID NO 20)

[0317] 5′ TTTCTAGATTAGGGCGTAGCGGGGGCGGGCG 3′.

[0318] This PCR fragment is then cloned into the plasmid pVR1012(Example 2) previously digested with PstI and XbaI, generating theplasmid pPB284, having a size of about 5943 bp. The truncated gD gene ofBHV-1 encodes a protein of 355 amino acids.

[0319] 3.3.3 pSB117: gD Gene (tPA Δ[TM−Cter] form) cloned into thevector pAB110

[0320] The tPA Δ[TM−Cter] form of the BHV-1 gD gene is amplified by PCRfrom the pPB284 template (Example 3.3.2) and with the aid of thefollowing primers:

[0321] SB225 (39 mer) (SEQ ID NO 21)

[0322] 5′ AAAATTTCGATATCCCCCGCGCCGCGGGTGACGGTATAC 3′ and

[0323] SB226 (33 mer) (SEQ ID NO 22)

[0324] 5′ GGAAGATCTTTAGGGCGTAGCGGGGGCGGGCGG 3′.

[0325] The amplification product (1029 bp) is digested with the enzymesEcoRV and BglII and cloned into the vector pAB110 (Example 2) previouslydigested with EcoRV and BglII, generating the plasmid pSB117, having asize of about 6071 bp.

[0326] The tPA Δ[TM−Cter] form of the gD gene encodes a glycoprotein of368 amino acids, containing the extracellular domain of the BHV-1 gDglycoprotein.

Example 4 Plasmids encoding the various forms of the bovine respiratorysencitial virus (BRSV) antigens

[0327] The genes encoding the F and G antigens of the BRSV virus areobtained by RT-PCR from the viral RNA of the Snook strain (Thomas et al.Research in Vet. Science, 1982, 33, 170-182). The BRSV A 51908 strain(ATCC number VR-794) may also be used.

[0328] 4.1 Plasmids Encoding the Various Forms of BRSV-F

[0329] 4.1.1 pSB107: F Gene (Native Form) Cloned Into the Vector pVR1012

[0330] The F gene of the Snook strain of BRSV is amplified by RT-PCRusing the viral RNA as template and with the aid of the followingprimers:

[0331] SB210 (34 mer) (SEQ ID NO 23)

[0332] 5′ AAATTTTCTGCAGATGGCGACAACAGCCATGAGG 3′ and

[0333] SB211 (35 mer) (SEQ ID NO 24)

[0334] 5′ TTAAGGATCCTCATTTACTAAAGGAAAGATTGTTG 3′.

[0335] The amplification product, having a size of 1739 bp, is digestedwith the enzymes PstI and BamHI and cloned into the vector pVR1012(Example 2) previously digested with PstI and BamHI, thus generating theplasmid pSB107, having a size of about 6583 bp.

[0336] The F gene of the BRSV virus encodes a protein of 574 aminoacids.

[0337] 4.1.2 pSB108: F gene (Δ[TM−Cter] form) cloned into the vectorpVR1012

[0338] The truncated form of the F gene of the Snook strain of BRSV isamplified by RT-PCR using the viral RNA as template and with the aid ofthe following primers:

[0339] SB210 (SEQ ID NO 23) and

[0340] SB212 (39 mer) (SEQ ID NO 25)

[0341] 5′ AATTTTGGATCCTCATGTGGTGGATTTTCCTACATCTAC 3′.

[0342] The amplification product (1581 bp) is digested with the enzymesPstI and BamHI and cloned into the vector pVR1012 (Example 2) previouslydigested with PstI and BamHI, generating the plasmid pSB108, having asize of about 6430 bp.

[0343] The truncated form of the F gene encodes a glycoprotein of 523amino acids, containing the extracellular domain of the BRSV Fglycoprotein.

[0344] 4.1.3 pSB114: F gene (tPA Δ[TM−Cter] form) cloned into the vectorpAB110

[0345] The tPA Δ[TM−Cter] form of the F gene of the BRSV Snook strain isamplified by RT-PCR using the viral RNA as template and with the aid ofthe following primers:

[0346] SB212 (SEQ ID NO 25) and

[0347] SB220 (38 mer) (SEQ ID NO 26)

[0348] 5′ AAAATTCACGTGAACATAACAGAAGAATTTTATCAATC 3′.

[0349] The amplification product (1516 bp) is digested with the enzymesPmlI and BglII and cloned into the vector pAB110 (Example 2) previouslydigested with PmlI and BglII, generating the plasmid pSB114, having asize of about 6572 bp.

[0350] The tPA Δ[TM−Cter] form of the F gene encodes a glycoprotein of535 amino acids, containing the extracellular domain of the BRSV Fglycoprotein.

[0351] 4.1.4 pPB449: F Gene (Large Deletion Δ[TM−Cter] Form) Cloned Intothe Vector pVR1012

[0352] The truncated form of the F gene of the Snook strain of BRSV isamplified by RT-PCR using the viral RNA as template and with the aid ofthe following primers:

[0353] SB210 (SEQ ID NO 23) and

[0354] FC129 (39 mer) (SEQ ID NO 57)

[0355] 5′ AATTTTGGATCCTCAGATTCCACGATTTTTATTAGAAGC 3′.

[0356] The amplification product (1305 bp) is digested with the enzymesPstI and BamHI and cloned into the vector pVR1012 (Example 2) previouslydigested with PstI and BamHI, generating the plasmid pPB449, having asize of about 6150 bp.

[0357] The truncated form of the F gene encodes a glycoprotein of 431amino acids, containing the extracellular domain of the BRSV Fglycoprotein.

[0358] 4.2 Plasmids Encoding the Various Forms of BRSV-G

[0359] In the case of the BRSV G protein (type II glycoprotein), thesignal sequence and the transmembrane sequence are indistinguishable,requiring the addition of a signal sequence upstream of the sequencecorresponding to the extracellular domain during the deletion of thetransmembrane domain.

[0360] The plasmid pAB110 (Example 2) is used for the construction ofthe plasmids containing the truncated forms of the gene encoding theBRSV G protein.

[0361] 4.2.1 pSB109: G Gene (Native Form) Cloned Into the Vector pVR1012

[0362] The G gene of the BRSV Snook strain is amplified by RT-PCR usingthe viral RNA as template and with the aid of the following primers:

[0363] SB213 (32 mer) (SEQ ID NO 27)

[0364] 5′ ACGCGTCGACATGTCCAACCATACCCATCATC 3′ and

[0365] SB214 (38 mer) (SEQ ID NO 28)

[0366] 5′ TTAAAATCTAGATTAGATCTGTGTAGTTGATTGATTTG 3′.

[0367] The amplification product (784 bp) is digested with enzymes SalIand XbaI and cloned into the vector pVR1012 (Example 2) previouslydigested with SalI and XbaI, generating the plasmid pSB109, having asize of about 5661 bp.

[0368] The BRSV G gene encodes a glycoprotein of 257 amino acids.

[0369] 4.2.2 pSB110: G gene (tPA Δ[TM−Cter] form) cloned into the vectorpAB110

[0370] The truncated form of the G gene of the BRSV Snook strain isamplified by RT-PCR using the viral RNA as template and with the aid ofthe following primers:

[0371] SB215 (33 mer) (SEQ ID NO 29)

[0372] 5′ TTTTAAGGATCCGCTAAAGCCAAGCCCACATCC 3′ and

[0373] SB216 (33 mer) (SEQ ID NO 30)

[0374] 5′ TTAAAATCTAGATTAGATCTGTGTAGTTGATTG 3′.

[0375] The amplification product (666 bp) is digested with the enzymesBamHI and XbaI and cloned into the vector pAB110 (Example 2) previouslydigested with BamHI and XbaI, generating the plasmid pSB110, having asize of about 5660 bp.

[0376] The tPA Δ[TM−Cter] form of the BRSV virus G gene encodes aglycoprotein of 218 amino acids, containing the extracellular domain ofthe G glycoprotein, but preceded by the signal sequence of the tissueplasminogen activator.

[0377] 4.3 pFC123: N Gene (Native Form) Cloned Into the Vector pVR1012

[0378] The N gene of BRSV is amplified by RT-PCR using the viral RNA astemplate and with the aid of the following primers:

[0379] FC130 (34 mer) (SEQ ID NO 58)

[0380] 5′ AAATTTTGTCGACATGGCTCTTAGCAAGGTCAAA 3′ and

[0381] FC131 (35 mer) (SEQ ID NO 59)

[0382] 5′ TTAAGGATCCTCACAGTTCCACATCATTGTCTTTG 3′.

[0383] The amplification product, having a size of 1199 bp, is digestedwith the enzymes SalI and BamHI and cloned into the vector pVR1012(Example 2) previously digested with SalI and BamHI, thus generating theplasmid pFC123, having a size of about 6057 bp.

[0384] The N gene of the BRSV virus encodes a nucleocapsid protein of391 amino acids.

Example 5 Plasmids Encoding the Various Forms of the Bovine ViralDiarrhea Virus Type 1 (BVD-1) Antigens

[0385] The genes encoding the E0 (glycoprotein of 48 kDa or gp48) and E2(gp53) antigens of the type I BVDV viruses are obtained by RT-PCR fromthe viral RNA of the Osloss strain (L. De Moerlooze et al. J. Gen.Virol. 1993, 74, 1433-1438; A. Renard et al., DNA, 1985, 4, 439-438; A.Renard et al. Ann. Rech. Vet., 1987, 18, 121-125). The NADL (ATCCVR-534) or New York (ATCC VR-524) strains may also be used.

[0386] 5.1 Plasmids Encoding the Various Forms of E0 of the BVDV Type 1Osloss Strain

[0387] 5.1.1 pLF1028: E0 Gene (Native Form) Cloned Into the VectorpVR1012

[0388] The complementary DNA (cDNA) of the E0 gene of the Osloss strainis synthesized from the corresponding viral RNA with the aid of theprimer LF051 and amplified by the PCR reaction with the aid of thefollowing oligonucleotide pair:

[0389] LF050 (36 mer) (SEQ ID NO 31)

[0390] 5′ CATACCGTCGACATGAAGAAACTAGAGAAAGCCCTG 3′ and

[0391] LF051 (40 mer) (SEQ ID NO 32)

[0392] 5′ CATACCGGATCCTCAGGCTGCATATGCCCCAAACCATGTC 3′.

[0393] The DNA fragment of about 765 bp obtained by digesting the PCRproduct with SalI and BamHI is ligated with a fragment of 4866 bpresulting from the digestion of pVR1012 (Example 2) with SalI and BamHIin order to generate the plasmid pLF1028 (about 5636 bp). The E0 gene ofBVDV-1 strain Osloss encodes a protein of 252 amino acids.

[0394] An ATG codon is introduced into the sequence of theoligonucleotide LF050 so as to allow the initiation of the translationof the corresponding recombinant E0 polypeptide.

[0395] 5.1.2 pLF1029: E0 Gene, (β-Globin tPA-E0) Form Cloned Into theVector pLF999.

[0396] The E0 gene is synthesized by a PCR reaction from the pLF1028template (Example 5.1.1) and with the aid of the followingoligonucleotide pair:

[0397] LF052 (39 mer) (SEQ ID NO 33)

[0398] 5′ CATGACGCGGCCGCTATGAAGAAACTAGAGAAAGCCCTG 3′ and

[0399] LF053 (40 mer) (SEQ ID NO 34)

[0400] 5′ CATGACAGATCTTTAGGCTGCATATGCCCCAAACCATGTC 3′.

[0401] The DNA fragment of about 770 bp obtained by digesting the PCRproduct with NotI and BglII is ligated with a fragment of 5642 bpresulting from the digestion of pLF999 (Example 2) with NotI and BglIIin order to generate the plasmid pLF1029 (about 6417 bp).

[0402] The E0 gene of BVDV-1 strain Osloss thus modified (β-globintPA-E0) encodes a protein of 283 amino acids.

[0403] 5.2 Plasmids Encoding the Various Forms of E2 of the BVDV Type 1Osloss Strain

[0404] 5.2.1 pLF1020: E2 Gene (Native Form) Cloned Into the VectorpVR1012

[0405] The cDNA of the E2 gene of the Osloss strain is synthesized fromthe corresponding viral RNA with the aid of the primer LF040 andamplified by a PCR reaction with the aid of the followingoligonucleotide pair:

[0406] LF039 (33 mer) (SEQ ID NO 35)

[0407] 5′ CATGACGTCGACATGACGACTACTGCATTCCTG 3′ and

[0408] LF040 (36 mer) (SEQ ID NO 36)

[0409] 5′ CATGACAGATCTTCAACGTCCCGAGGTCATTTGTTC 3′.

[0410] The DNA fragment of 1235 bp obtained by digesting the PCR productwith SalI and BglII is ligated with a fragment of 4860 bp resulting fromthe digestion of pVR0112 (Example 2) with SalI and BglII in order togenerate the plasmid pLF1020 (about 6100 pb).

[0411] The E2 gene of BVDV-1 strain Osloss encodes a protein of 409amino acids.

[0412] An ATG codon is introduced into the sequence of theoligonucleotide LF039 so as to allow the initiation of the translationof the corresponding recombinant E2 polypeptide.

[0413] 5.2.2 pLF1021: E2 Gene, (β-globin tPA-E2 Δ[TM+Cter]) form ClonedInto the Vector pLF999.

[0414] The E2 gene deleted for its transmembrane and carboxy-terminaldomains is synthesized by a PCR reaction from the pLF1020 template(Example 5.2.1) and with the aid of the following oligonucleotide pair:

[0415] LF041 (36 mer) (SEQ ID NO 37)

[0416] 5′ CATGACGCGGCCGCTATGACGACTACTGCATTCCTG 3′ and

[0417] LF042 (35 mer) (SEQ ID NO 38)

[0418] 5′ CATGACAGATCTCAAGCGAAGTAATCCCGGTGGTG 3.

[0419] The DNA fragment of 1132 bp obtained by digesting the PCR productwith NotI and BglII is ligated with a fragment of 5642 bp resulting fromthe digestion of pLF999 (Example 2) with NotI and BglII in order togenerate the plasmid pLF1021 (about 6779 bp).

[0420] The E2 gene of BVDV-1 strain Osloss thus modified (β-globintPA-E2 Δ[TM+Cter]) encodes a protein of 404 amino acids.

Example 6 Plasmids Encoding the Various Forms of the Bovine ViralDiarrhea Virus Type 2 (BVDV-2) Antigens

[0421] The genes encoding the E2 antigen (gp53) of the BVDV type 2viruses are obtained by RT-PCR from the viral RNA of the strain 890 (J.F. Ridpath and S. R. Bolin, Virology, 1995, 212, 36-46). The strain Q140can also be used and may be obtained from the Quebec Ministry ofAgriculture, Fisheries and Food, Armand-Frappier Institute (P. Tijssenet al., Virology, 1996, 217, 356-361). The strains 1373 and 296 may alsobe used (J. F. Ridpath, BVDV Research Project, National Animal DiseaseCenter, 2300 Dayton Avenue, Ames, USA).

[0422] 6.1 Plasmids Encoding the Various Forms of E2 of the Type 2-890Strain

[0423] 6.1.1. pLF1022: E2 Gene (Native Form) Cloned Into the VectorpVR1012

[0424] The cDNA of the E2 gene of the strain 890 is synthesized from thecorresponding viral RNA with the aid of the primer LF044 amplified by aPCR reaction with the aid of the following oligonucleotide pair:

[0425] LF043 (36 mer) (SEQ ID NO 39)

[0426] 5′ ACTGTATCTAGAATGACCACCACAGCTTTCCTAATC 3′ and

[0427] LF044 (39 mer) (SEQ ID NO 40)

[0428] 5′ ACTGTAAGATCTTTAAGTATTCACTCCAGCACCCATAGC 3′.

[0429] The DNA fragment of about 1240 bp obtained by digesting the PCRproduct with XbaI and BglII is ligated with a fragment of 4891 bpresulting from the digestion of pVR1012 (Example 2) with XbaI and BglIIin order to generate the plasmid pLF 1022 (about 6136 bp).

[0430] The E2 gene of BVDV-2 strain 890 encodes a protein of 410 aminoacids.

[0431] An ATG codon is introduced into the sequence of theoligonucleotide LF043 so as to allow the initiation of the translationof the corresponding recombinant E2 polypeptide.

[0432] 6.1.2 pLF1023: E2 gene, (β-globin tPA-E2 Δ[TM+Cter]) form, clonedinto the vector pLF999

[0433] The E2 gene deleted for its transmembrane and carboxy-terminaldomains is synthesized by a PCR reaction from the pLF1022 template(Example 6.2.1) and with the aid of the following oligonucleotide pair:

[0434] LF045 (41 mer) (SEQ ID NO 41)

[0435] 5′CATGACGCGGCCGCCCTATGACCACCACAGCTTTCCTAATC 3′ and

[0436] LF046 (36 mer) (SEQ ID NO 42)

[0437] 5′ CATGACAGATCTTTATATGAACTCTGAGAAGTAGTC 3′.

[0438] The DNA fragment of about 1140 bp obtained by digesting the PCRproduct with NotI and BglII is ligated with a fragment of 5642 bpresulting from the digestion of pLF999 (Example 2) with NotI and BglIIin order to generate the plasmid pLF1023 (about 6787 bp).

[0439] The E2 gene of BVDV-2 strain 890 thus modified (β-globin tPA-E2Δ[TM+Cter]) encodes a protein of 405 amino acids.

[0440] 6.2 Plasmids Encoding the Various Forms of E0 of the Type 2-890Strain 6.2.1 pLF 1030: E0 gene (native form) cloned into the vectorpVR1012

[0441] The cDNA of the E0 gene of the 890 strain is synthesized from thecorresponding viral RNA with the aid of the LF065 primer and amplifiedby a PCR reaction with the aid of the following oligonucleotide pair:

[0442] LF064 (39 mer) (SEQ ID NO 43)

[0443] 5′ CATACCGTCGACATGAGAAAGAAATTGGAGAAGGCACTG 3′ and

[0444] LF065 (39 mer) (SEQ ID NO 44)

[0445] 5′ CATACCGGATCCTCATGCTGCATGAGCACCAAACCATGC 3′.

[0446] The DNA fragment of about 768 bp obtained by digesting the PCRproduct with SalI and BamHI is ligated with a fragment of 4866 bpresulting from the digestion of pVR0112 (Example 2) with SalI and BamHIin order to generate the plasmid pLF1030 (about 5639 bp). The E0 gene ofBVDV-2 strain 890 encodes a protein of 253 amino acids.

[0447] An ATG codon is introduced into the sequence of theoligonucleotide LF064 so as to allow the initiation of the translationof the corresponding recombinant E0 polypeptide.

[0448] 6.2.2 pLF1031: E0 gene, (β-globin tPA-E0) form, cloned into thevector pLF999.

[0449] The E0 gene is synthesized by a PCR reaction from the pLF1030template (Example 6.2.1.) and with the aid of the followingoligonucleotide pair:

[0450] LF066 (42 mer) (SEQ ID NO 45)

[0451] 5′ CATGACGCGGCCGCTATGAGAAAGAAATTGGAGAAGGCACTG 3′ and

[0452] LF067 (39 mer) (SEQ ID NO 46)

[0453] 5′ CATACCAGATCTTCATGCTGCATGAGCACCAAACCATGC 3′.

[0454] The DNA fragment of about 770 bp obtained by digesting the PCRproduct with NotI and BglII is ligated with a fragment of 5642 bpresulting from the digestion of pLF999 (Example 2) with NotI and BglIIin order to generate the plasmid pLF1031 (about 6417 bp).

[0455] The E0 gene of BVDV-2 strain 890 thus modified (β-globin tPA-E0)encodes a protein of 283 amino acids.

Example 7 Plasmids Encoding the Various Forms of the BovineParainfluenza Virus Type 3 (bPI-3) Antigens

[0456] The genes encoding the hemagglutinin-neuraminidase (HN) andfusion (F) antigens of the bPI-3 virus are obtained by RT-PCR from theviral RNA of the Reisinger SF-4 strain (accessible from ATCC under thenumber VR-281).

[0457] 7.1 Plasmids Encoding the Various Forms of HN of the bPI-3 SF-4Strain

[0458] 7.1.1 pLF1024: HN Gene (Native Form) Cloned Into the VectorpVR1012

[0459] The cDNA of the HN gene of the SF-4 strain is synthesized fromthe corresponding viral RNA with the aid of the primer LF048 andamplified by a PCR reaction with the aid of the followingoligonucleotide pair:

[0460] LF047 (39 mer) (SEQ ID NO 47)

[0461] 5′ CATATCGTCGACATGGAATATTGGAAACACACAAACAGC 3′ and

[0462] LF048 (38 mer) (SEQ ID NO 48)

[0463] 5′ CATGACGATATCTAGCTGCAGTTTTTCGGAACTTCTGT 3′.

[0464] The DNA fragment of 1726 bp obtained by digesting the PCR productwith SalI and EcoRV is ligated with a fragment of 4896 bp resulting fromthe digestion of pVRO112 (Example 2) with SalI and EcoRV in order togenerate the plasmid pLF1024 (about 6619 bp).

[0465] The bPI-3 HN gene encodes a protein of 572 amino acids.

[0466] 7.1.2 pLF1025: HN gene, (β-globin tPA-E2 Δ[TM]) form, cloned intothe vector pLF999

[0467] The HN gene deleted for its transmembrane domain is synthesizedby a PCR reaction from the pLF1024 template (Example 7.1.1) with the aidof the following oligonucleotide pair:

[0468] LF058 (33 mer) (SEQ ID NO 49)

[0469] 5′ CATACTGCGGCCGCTTTAATTCAAGAGAACAAT 3′ and

[0470] LF059 (35 mer) (SEQ ID NO 50)

[0471] 5′ CATATCGATATCTAGCTGCAGTTTTTCGGAACTTC 3′.

[0472] The DNA fragment of 1566 bp obtained by digesting the PCR productwith NotI and EcoRV is ligated with a fragment of 5663 bp resulting fromthe digestion of pLF999 (Example 2) with NotI and EcoRV in order togenerate the plasmid pLF1025 (about 7229 bp).

[0473] The bPI-3 HN gene thus modified (β-globin tPA-E2 Δ[TM]) encodes aprotein of 548 amino acids.

[0474] 7.2 Plasmids encoding the various forms of F of the bPI-3 SF-4strain

[0475] 7.2.1 pLF1026: F Gene (Native Form) Cloned Into the VectorpVR1012

[0476] The cDNA of the F gene of strain SF-4 is synthesized from thecorresponding viral RNA with the aid of the primer LF061 and amplifiedby a PCR reaction with the aid of the following oligonucleotide pair:

[0477] LF060 (36 mer) (SEQ ID NO 51)

[0478] 5′ CATATCGTCGACATGATCATCACAAACACAATCATA 3′ and

[0479] LF061 (36 mer) (SEQ ID NO 52)

[0480] 5′ CATGACCAGATCTTATTGTCTATTTGTCAGTATATA 3′.

[0481] The DNA fragment of 1628 bp obtained by digesting the PCR productwith SalI and BglII is ligated with a fragment of 4860 bp resulting fromthe digestion of pVRO112 (Example 2) with SalI and BglII in order togenerate the plasmid pLF1026 (about 6488 bp).

[0482] The bPI-3 F gene encodes a protein of 550 amino acids.

[0483] 7.2.2 pLF1027: F Gene, (β-globin tPA-F Δ[TM+Cter]) Form, ClonedInto the Vector pLF999

[0484] The F gene deleted for its transmembrane and C-terminal domainsis synthesized by a PCR reaction from the pLR1026 template (Example7.2.1) and with the aid of the following oligonucleotide pair:

[0485] LF062 (42 mer) (SEQ ID NO 53)

[0486] 5′ CATACTGCGGCCGCTCAAATAGACATAACAAAACTGCAACGT 3′ and

[0487] LF063 (41 mer) (SEQ ID NO 54)

[0488] 5′ CATATCGATATCTATGCACTAGATTGATACCAACTTCCAAC 3′.

[0489] The DNA fragment of 1434 bp obtained by digesting the PCR productwith NotI and EcoRV is ligated with a fragment of 5663 bp resulting fromthe digestion of pLF999 (Example 2) with NotI and EcoRV in order togenerate the plasmid pLF1027 (about 7097 bp).

[0490] The bPI-3 F gene thus modified (β-globin tPA-F Δ[TM+Cter])encodes a protein of 504 amino acids.

Example 8 Plasmid encoding bovine GM-CSF

[0491] The cDNA of the bovine GM-CSF gene is synthesized from thecellular RNA of bovine blood mononucleated cells with the aid of theprimer LF065 and amplified by a PCR reaction with the aid of thefollowing oligonucleotide pair:

[0492] LF054 (36 mer) (SEQ ID NO 55)

[0493] 5′ CATATCGTCGACATGTGGCTGCAGAACCTGCTTCTC 3′ and

[0494] LF055 (34 mer) (SEQ ID NO 56)

[0495] 5′ CATGACCAGATCTTCACTTCTGGGCTGGTTCCCA 3′.

[0496] The DNA fragment of 437 bp obtained by digesting the PCR productwith SalI and BglII is ligated with a fragment of 4860 bp resulting fromthe digestion of pVR1012 (Example 2) with SalI and BglII in order togenerate the plasmid pLF1032 (about 5297 bp). The bovine GM-CSF geneencodes a protein of 143 amino acids.

Example 9 Formulation of the Vaccinal Plasmids

[0497] The DNA solution containing one or more plasmids according toExamples 3 to 8 is concentrated by ethanolic precipitation as describedin Sambrook et al. (1989). The DNA pellet is taken up in a 0.9% NaClsolution so as to obtain a concentration of 1 mg/ml. A 0.75 mMDMRIE-DOPE solution is prepared by taking up a lyophilisate ofDMRIE-DOPE with an appropriate volume of sterile H₂O.

[0498] The formation of the plasmid DNA-lipid complexes is achieved bydiluting, in equal parts, the 0.75 mM DMRIE-DOPE solution with the DNAsolution at 1 mg/ml in 0.9% NaCl. The DNA solution is graduallyintroduced, with the aid of a seamed 26G needle, along the wall of thevial containing the cationic lipid solution so as to avoid the formationof foam. Gentle shaking is carried out as soon as the two solutions havebeen mixed. A composition comprising 0.375 mM of DMRIE-DOPE and 500μg/ml of plasmid is finally obtained.

[0499] It is desirable for all the solutions used to be at roomtemperature for all the operations described above. The DNA/DMRIE-DOPEcomplex formation is allowed to take place at room temperature for 30minutes before immunizing the animals.

Example 10 Immunization of Bovines Against BHV-1

[0500] 12 bovines are randomized into 3 groups of 4 s.

[0501] Group 1 constitutes the control animal group.

[0502] A mixture of vaccinal plasmids pPB281 (encoding BHV-1 gB in aΔ[TM−Cter] form, Example 3.1.2), pPB292 (encoding BHV-1 gC in aΔ[TM−Cter] form, Example 3.2.2) and pPB284 (encoding BHV-1 gD in aΔ[TM−Cter] form, Example 3.3.2) is administered to the animals of Group2.

[0503] The same mixture as that in Group 2, but formulated withDMRIE-DOPE as is described in Example 15, is administered to the animalsof Group 3.

[0504] An injection of 10 ml, by the intramuscular route, is performedon each bovine with the aid of syringes equipped with needle, and isrepeated 21 days later. The total mass of each plasmid used during eachimmunization is 1500 μg.

[0505] Persons skilled in the art possess the necessary competence toadjust the volume or the concentration according to the plasmid doserequired.

[0506] Monitoring of the serological response induced by the twomixtures of vaccine plasmids expressing the BHV-1 gB, gC and gD antigensis carried out over a period of 35 days after the first vaccination.

[0507] The results are presented in the table which follows: Formu-Plasmids lation Antigens Dose SN at D28 SN at D35 Control — — — 0.2 +/−0.0 0.2 +/− 0.0 pPB281 — gB Δ[TM-Cter] 1500 1.0 +/− 0.5 1.2 +/− μg 0.8pPB292 gC Δ[TM-Cter] 1500 μg pPB294 gD Δ[TM-Cter] 1500 μg pPB281 DMRIE-gB Δ[TM-Cter] 1500 2.1 +/− 0.6 2.7 +/− DOPE μg 0.6 pPB292 gC Δ[TM-Cter]1500 μg pPB294 gD Δ[TM-Cter] 1500 μg

Example 11 Prime-Boost Immunization of Bovines Against BRSV

[0508] The vaccinal plasmids, encoding BRSV F in a large deletionΔ[TM−Cter] form and in a Δ[TM−Cter] form and encoding BRSV N, wereconcentrated by ethanolic precipitation as described in Sambrook et al.(1989). The DNA pellet was taken up in a 1.8% NaCl solution so as toobtain a concentration of 1.6 mg/ml. A 1.2 mM DMRIE-DOPE solution wasprepared by taking up a lyophilisate of DMRIE-DOPE with an appropriatevolume of sterile H₂O.

[0509] The formation of the plasmid DNA-lipid complexes was achieved bymixing, in equal parts, the 1.2 mM DMRIE-DOPE solution with the DNAsolution at 1.6 mg/ml in 1.8% NaCl. The DNA solution was graduallyintroduced, with the aid of a seamed 26G needle, along the wall of thevial containing the cationic lipid solution so as to avoid the formationof foam. Gentle shaking was carried out as soon as the two solutionshave been mixed. A composition comprising 0.6 mM of DMRIE-DOPE and 800μg/ml of plasmid was finally obtained.

[0510] It is desirable for all the solutions used to be at roomtemperature for all the operations described above. The DNA/DMRIE-DOPEcomplex formation is allowed to take place at room temperature for 30minutes before immunizing the animals.

[0511] BRSV (strain 375, deposited before the American Type CultureCollection (ATCC) under the accession number # VR-1339) was inactivatedwith β-propiolactone and formulated with Carbopol® 974P (Noveon Inc.)(20% v/v, 15 g/L Carbopol solution) to have a 3 mg/ml finalconcentration of Carbopol. 20 bovines, 3 to 4 weeks old, were randomizedinto 4 groups of 5 animals.

[0512] Group 1 constitutes the control animal group (without anyvaccination).

[0513] For the priming immunization, DNA vaccine formulated withDMRIE-DOPE was administered to the animals of Group 2 and Group 3 by theintramuscular route, 2 ml per dose, with a syringe and a needle. Thetotal quantity of plasmids used for the immunization was 1600 μg.Inactivated vaccine formulated with Carbopol was administered to theanimals of Group 4 by the intramuscular route, 5 ml per dose, with asyringe and a needle. 28 days later, a boost immunization was done. DNAvaccine formulated with DMRIE-DOPE was administered to the animals ofGroup 2 by the intramuscular route, 2 ml per dose, with a syringe and aneedle. The total quantity of plasmids used for the immunization was1600 μg. Inactivated vaccine formulated with Carbopol was administeredto the animals of Group 3 and Group 4 by the intramuscular route, 5 mlper dose, with a syringe and a needle.

[0514] All the animals were challenged on day 193 with pathogenic BRSV(Snook strain; Taylor G. et al., J Gen Virol 1998, 79(7), 1759-67) atabout 4.5 log10 CCID₅₀/ml. 10 ml of the inoculum suspension wereinoculated intra-nasally to each of the calves by spray for a totalinfectious dose per calve of approximately 10⁵⁵ CCID₅₀.

[0515] Monitoring of the rectal temperatures, respiratory rates,clinical scores, lung lesion scores, viral excretions and memoryBRSV-specific IFN_(γ)+ T cell response was carried out over a period of10 days after the challenge.

[0516] The rectal temperatures results are presented in ° C. in the FIG.3.

[0517] All calves presented hyperthermia after challenge. Group 3(DNA/inactivated) presented an average temperature peak earlier in timethan the other group and rectal temperature were significantly lower(p=0.001) as compared to the control for the D4-D1O period.

[0518] The respiratory rates (breathing per minute) results arepresented in the FIG. 4.

[0519] Group 3 presented on average a lower and earlier respiratory ratepeak than the other groups. For this group, a lower respiratory rate wasobserved (p=0.06) when compared to the controls.

[0520] The clinical scores results are scored as followed: Clinical sign0 1 2 Prostation No Yes — Anorexia None Partial Total Coughing Noneoccasionnaly Repeted Dyspnea None Moderate Hard

[0521] The score for mortality or for euthanasia for ethical reason is8.

[0522] The clinical scores results are presented in the FIG. 5.

[0523] All calves presented moderate to severe hyperpnoea afterchallenge. All calves were at least prostrated or partially anorecticone days during the challenge phase. There were considerable variationin the severity of signs, even within groups. For example, in the group4, one animal was severely affected (deceased on D8) and its globalclinical score (GSS=total score of the challenge phase) account for morethan 50% of the total GSS for the group. The situation is even morestriking in the group 3. Group where GSS of one calve represent nearly2/3 of the total GSS for the group 3.

[0524] All calves (found dead or euthanazied) were necropsied. Amacroscopic examination of the deep respiratory apparatus was performedand the dorsal and the ventral sides of the lung were observed for lunglesions. Size of lesions was estimated for each pulmonary lobe (bothsides), as a percentage of lobe affected (surface affected/totalsurface), to calculate a lobe score as follow:

Lobe score=(lobe balance index)×(dorsal score +ventral score)/2

[0525] The lobe balance index represents the relative importance ofpulmonary lobes and is:

[0526] For the right cranial lobe and the medium cranial lobe: 0.11

[0527] For the right medium caudal lobe: 0.07

[0528] For the right caudal lobe: 0.35

[0529] For the left cranial lobe: 0.05

[0530] For the left medium lobe: 0.06

[0531] For the left caudal lobe: 0.32

[0532] And for the azygos lobe: 0.04.

[0533] The lung lesion score of each calve was determined by addition ofall lobe scores.

[0534] The lung lesion scores results are presented in the FIG. 6.

[0535] Of particular interest, in the group 3, 4/5 calves had less than7.5% lung lesions whereas the remaining calve had almost 100% lunglesions.

[0536] The nasal swabs viral excretion results are presented in the FIG.7.

[0537] BRSV challenge strain was detected in animals of all groups,however at a variable level, controls having the highest level ofexcretion. As compared to controls, the total excretion (sum of dailyexcretion title in log₁₀) was significantly reduced in the group 3 only.

[0538] In vivo-primed bovine peripheral blood mononuclear cells (PBMCs)were taken from blood samples collected at day 36 and 92 followingvaccination and 10 days post-challenge.

[0539] Day 36 PBMCs were re-stimulated ex vivo with autologous dendriticcells infected either with a recombinant poxvirus expressing the Fprotein of BRSV or with the parental poxvirus as control.

[0540] Day 92 and 10 days post-challenge PBMCs were re-stimulated exvivo directly with live BRSV grown on VERO cells (strain 375, from about6.0 log₁₀ CCID₅₀/ml to about 6.4 log₁₀ CCID₅₀/ml). As control, PBMCswere mock infected by a VERO cell lysate.

[0541] The frequencies of antigen-specific T cells was determined by thenumber of IFN_(γ)-secreting cells in a quantitative enzyme-linked immunespot (ELISPOT) assay (Laval F. et al., Vet. Immunol. Immunopathol.,2002, 90(3-4), 191-201). Using this approach the vaccines strategies canbe rank for their ability to prime viral-specific IFNy(+) T cells.

[0542] The memory BRSV-specific IFN_(γ)+ T cell responses are presentedin the FIG. 8.

[0543] In contrast to the day 36 time point, where the resultstranslated into the secondary effector T cell response, day 92 analysisrevealed the memory T cell response (2 months following the secondshot). It appeared that calves with a good level of memory specific Tcell response were found mainly in the group 3 (calves vaccinated withthe DNA/inactivated prime-boost).

[0544] Interestingly, the calves of the group 3 which consistentlyshowed a memory BRSV-specific IFN_(γ)+ T cell response, whatever thetime point analysed, are the calves where a significant protection wasobserved.

[0545] The invention will be further described by the following numberedparagraphs:

[0546] 1. Use of a plasmid containing and expressing in vivo in a bovinesuch as cattle at least one immunogen from a bovine pathogen, selectedfrom BRSV, bPI-3, BHV-1 and BVDV, for the preparation of a DNA vaccineintended to induce an immune response into young bovines such as calveswhich have or may have maternal antibodies against said bovine pathogen.

[0547] 2. Use according to paragraph 1, wherein the DNA vaccine isintended to be administered to the young animal or bovine from calvingup to and including 12 weeks of age, such as from calving up to andincluding 6 weeks of age, such as from calving up to and including 4weeks of age, and especially from calving up to and including 3 weeks ofage.

[0548] 3. Use according to paragraph 1 or 2, wherein said DNA vaccine isintended to induce a priming immune response, such as with a IFN_(γ)+memory T cell response specific for the expressed immunogen, whichpriming immune response can be boosted by a subsequent administration ofan inactivated vaccine or a live recombinant vaccine comprising a viralvector, such as a live recombinant poxvirus, containing and expressingin vivo at least the same immunogen(s) than that expressed by the DNAvaccine.

[0549] 4. Use of a bovine pathogenic agent, selected from BRSV, bPI-3,BHV-1 and BVDV, for the preparation of a priming DNA vaccine comprisinga plasmid containing and expressing in vivo in a bovine such as cattleat least one immunogen from said pathogenic agent, and for thepreparation of a second vaccine comprising said pathogenic agent underan inactivated form, wherein the DNA vaccine is intended to beadministered to a bovine such as cattle first, such as to a young calvewhich have or may have maternal antibodies against said bovine pathogen,and the inactivated vaccine is intended to be administered after the DNAvaccine and to the same bovine such as cattle such as the young calve,to boost the immune response against said immunogen.

[0550] 5. Use according to paragraph 4, wherein the DNA vaccine isintended to induce in the bovine such as cattle, such as the calve, animmune response against said immunogen(s), such as the gamma+interferonmemory T cell response specific for the expressed immunogen.

[0551] 6. Use according to paragraph 4 or 5, wherein the DNA vaccine isintended to be administered to the young animal or bovine from calvingup to and including 12 weeks of age, such as from calving up to andincluding 6 weeks of age, such as from calving up to and including 4weeks of age, and especially from calving up to and including 3 weeks ofage.

[0552] 7. Use according to paragraph 4, 5 or 6, wherein the inactivatedvaccine is intended to be administered from about 2 weeks to about 5months after the priming administration, such as from about 3 to 6 weeksafter, and such as about 4 weeks after the DNA vaccine was administered.

[0553] 8. Use of a nucleotide sequence coding for at least one immunogenfrom a bovine pathogenic agent, selected from BRSV, bPI-3, BHV-1 andBVDV, for the preparation of a priming DNA vaccine comprising a plasmidcontaining and expressing in vivo said immunogen and for the preparationof a second vaccine comprising a live recombinant viral vector, such asa live recombinant poxvirus, containing and expressing in vivo at leastsaid immunogen(s), wherein the DNA vaccine is intended to beadministered to a bovine first, such as to a young calve which have ormay have maternal antibodies against said bovine pathogen, and the viralvector-based vaccine is intended to be administered after the DNAvaccine and to the same bovine such as the calve, to boost the immuneresponse against said immunogen.

[0554] 9. Use according to paragraph 8, wherein the DNA vaccine isintended to induce a DNA vaccine induced immune response against saidimmunogen(s), such as the IFN_(γ)+ memory T cell response specific forthe expressed immunogen.

[0555] 10. Use according to paragraph 8 or 9, wherein the DNA vaccine isintended to be administered to the young animal or bovine from calvingup to and including 12 weeks of age, such as from calving up to andincluding 6 weeks of age, such as from calving up to and including 4weeks of age, and especially from calving up to and including 3 weeks ofage.

[0556] 11. Use according to paragraph 8, 9 or 10, wherein the live viralvector-based vaccine is intended to be administered from about 2 weeksto about 5 months after the priming administration, such as from about 3to 6 weeks after, and such as about 4 weeks after the DNA vaccine wasadministered.

[0557] 12. Use of a bovine pathogenic agent, selected from BRSV, bPI-3,BHV-1 and BVDV, to prepare an inactivated vaccine intended to vaccinatea bovine against said pathogenic agent, wherein the bovine, such as ayoung calve which have or may have maternal antibodies against saidbovine pathogen, has previously been immunized with a DNA vaccineexpressing in vivo at least one immunogen from the same pathogenic agentand has developed a specific priming DNA vaccine induced immuneresponse, such as the IFN_(γ)+ memory T cell response specific for theexpressed immunogen.

[0558] 13. Use according to paragraph 12, wherein the inactivatedvaccine is intended to be administered from about 2 weeks to about 5months, such as from about 3 to 6 weeks, and such as about 4 weeks afterthe bovine was administered with the DNA vaccine.

[0559] 14. Use of a recombinant viral vector, such as a poxvirus vector,comprising and expressing in vivo at least one nucleotide sequencecoding for at least one immunogen from a bovine pathogenic agent,selected from BRSV, bPI-3, BHV-1 and BVDV, to prepare a live recombinantvaccine intended to vaccinate a bovine against the pathogenic agent,wherein the bovine, such as a young calve which have or may havematernal antibodies against said bovine pathogen, has previously beenimmunized with a DNA vaccine expressing in vivo at least the sameimmunogen(s), has developed a priming DNA vaccine induced immuneresponse, such as the IFN_(γ)+ memory T cell response specific for theexpressed immunogen.

[0560] 15. Use according to paragraph 14, wherein the live viralvector-based vaccine is intended to be administered about 2 weeks toabout 5 months, such as from about 3 to 6 weeks, and such as about 4weeks after the bovine was administered with the DNA vaccine.

[0561] 16. Prime-boost vaccination method of a bovine such as cattleagainst at least one bovine pathogen, wherein the bovine or cattle isfirst administered with a priming DNA vaccine comprising and expressingin vivo an immunogen from said pathogen, and then is boosted with asecond type of vaccine presenting the same immunogen.

[0562] 17. Method according to paragraph 16, wherein the boost is donewith an inactivated vaccine.

[0563] 18. Method according to paragraph 16, wherein the boost is donewith a vaccine comprising a recombinant live viral vector, such as arecombinant poxvirus, comprising and expressing in vivo the saidimmunogen.

[0564] 19. Method according to any one of paragraph 16 to 18, whereinthe DNA vaccine is administered to a young calve that can have maternalantibodies against the pathogenic agent against which immunization orvaccination is directed.

[0565] 20. Method according to any one of paragraph 16 to 19, whereinthe DNA vaccine is administered to the young animal or bovine fromcalving up to and including 12 weeks of age, such as from calving up toand including 6 weeks of age, such as from calving up to and including 4weeks of age, and especially from calving up to and including 3 weeks ofage.

[0566] 21. Method according to any one of paragraphs 16 to 20, whereinthe boost administration is administered from about 2 weeks to about 5months after the priming administration, such as from about 3 to 6 weeksafter, and such as about 4 weeks after.

[0567] 22. Method according to any one of paragraphs 16 to 21, wherein asecond administration of the boost vaccine is done, such as when thebovine or calves are transferred to the finishing units.

[0568] 23. The prime-boost vaccination method of a cattle against atleast one bovine pathogen, which comprises administering first to abovine such as cattle a priming DNA vaccine comprising a nucleotidesequence encoding and expressing in vivo an immunogen from saidpathogen, and then administering a second type of vaccine presenting thesame immunogen, e.g., an inactivated, attenuated, subunit, orrecombinant live viral vector vaccine, advantageously an inactivatedvaccine or a recombinant live viral vector, such as a recombinantpoxvirus, comprising and expressing in vivo the said immunogen. Themethod can be applied to a young calve that can have maternal antibodiesagainst the pathogenic agent against which immunization or vaccinationis directed.

[0569] Having thus described in detail preferred embodiments of thepresent invention, it is to be understood that the invention defined bythe appended claims is not to be limited to particular details set forthin the above description as many apparent variations thereof arepossible without departing from the spirit or scope of the presentinvention.

1 59 1 40 DNA Artificial Sequence Description of Artificial SequenceSynthetic oligonucleotide 1 gatctgcagc acgtgtctag aggatatcga attcgcggcc40 2 40 DNA Artificial Sequence Description of Artificial SequenceSynthetic oligonucleotide 2 gatccgcggc cgcgaattcg atatcctcta gacacgtgct40 3 20 DNA Artificial Sequence Description of Artificial SequenceSynthetic oligonucleotide 3 ttggggaccc ttgattgttc 20 4 21 DNA ArtificialSequence Description of Artificial Sequence Synthetic oligonucleotide 4ctgtaggaaa aagaagaagg c 21 5 30 DNA Artificial Sequence Description ofArtificial Sequence Synthetic oligonucleotide 5 ctccatgtcg acttggggacccttgattgt 30 6 30 DNA Artificial Sequence Description of ArtificialSequence Synthetic oligonucleotide 6 ctccatgtcg acctgtagga aaaagaagaa 307 30 DNA Artificial Sequence Description of Artificial SequenceSynthetic oligonucleotide 7 ttgtcgacat ggccgctcgc ggcggtgctg 30 8 21 DNAArtificial Sequence Description of Artificial Sequence Syntheticoligonucleotide 8 gcagggcagc ggctagcgcg g 21 9 52 DNA ArtificialSequence Description of Artificial Sequence Synthetic oligonucleotide 9ctgcacgagc tccggttcta cgacattgac cgcgtggtca agacggactg ag 52 10 56 DNAArtificial Sequence Description of Artificial Sequence Syntheticoligonucleotide 10 gatcctcagt ccgtcttgac cacgcggtca atgtcgtagaaccggagctc gtgcag 56 11 39 DNA Artificial Sequence Description ofArtificial Sequence Primer 11 aaaatttcga tatccgccgc ggggcgaccg gcgacaacg39 12 33 DNA Artificial Sequence Description of Artificial SequencePrimer 12 ggaagatctt cagtccgtct tgaccacgcg gtc 33 13 37 DNA ArtificialSequence Description of Artificial Sequence Synthetic oligonucleotide 13tcgtgcctgc ggcgcaaggc ccgggcgcgc ctgtagt 37 14 37 DNA ArtificialSequence Description of Artificial Sequence Synthetic oligonucleotide 14ctagactaca ggcgcgcccg ggccttgcgc cgcaggc 37 15 43 DNA ArtificialSequence Description of Artificial Sequence Synthetic oligonucleotide 15gcaccgctgc ccgagttctc cgcgaccgcc acgtacgact agt 43 16 43 DNA ArtificialSequence Description of Artificial Sequence Synthetic oligonucleotide 16ctagactagt cgtacgtggc ggtcgcggag aactcgggca gcg 43 17 39 DNA ArtificialSequence Description of Artificial Sequence Primer 17 aaaatttcgatatcccggcg ggggctcgcc gaggaggcg 39 18 32 DNA Artificial SequenceDescription of Artificial Sequence Primer 18 ggaagatctc tagtcgtacgtggcggtcgc gg 32 19 33 DNA Artificial Sequence Description of ArtificialSequence Primer 19 tttctgcaga tgcaagggcc gacattggcc gtg 33 20 31 DNAArtificial Sequence Description of Artificial Sequence Primer 20tttctagatt agggcgtagc gggggcgggc g 31 21 39 DNA Artificial SequenceDescription of Artificial Sequence Primer 21 aaaatttcga tatcccccgcgccgcgggtg acggtatac 39 22 33 DNA Artificial Sequence Description ofArtificial Sequence Primer 22 ggaagatctt tagggcgtag cgggggcggg cgg 33 2334 DNA Artificial Sequence Description of Artificial Sequence Primer 23aaattttctg cagatggcga caacagccat gagg 34 24 35 DNA Artificial SequenceDescription of Artificial Sequence Primer 24 ttaaggatcc tcatttactaaaggaaagat tgttg 35 25 39 DNA Artificial Sequence Description ofArtificial Sequence Primer 25 aattttggat cctcatgtgg tggattttcc tacatctac39 26 38 DNA Artificial Sequence Description of Artificial SequencePrimer 26 aaaattcacg tgaacataac agaagaattt tatcaatc 38 27 32 DNAArtificial Sequence Description of Artificial Sequence Primer 27acgcgtcgac atgtccaacc atacccatca tc 32 28 38 DNA Artificial SequenceDescription of Artificial Sequence Primer 28 ttaaaatcta gattagatctgtgtagttga ttgatttg 38 29 33 DNA Artificial Sequence Description ofArtificial Sequence Primer 29 ttttaaggat ccgctaaagc caagcccaca tcc 33 3033 DNA Artificial Sequence Description of Artificial Sequence Primer 30ttaaaatcta gattagatct gtgtagttga ttg 33 31 36 DNA Artificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 31cataccgtcg acatgaagaa actagagaaa gccctg 36 32 40 DNA Artificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 32cataccggat cctcaggctg catatgcccc aaaccatgtc 40 33 39 DNA ArtificialSequence Description of Artificial Sequence Synthetic oligonucleotide 33catgacgcgg ccgctatgaa gaaactagag aaagccctg 39 34 40 DNA ArtificialSequence Description of Artificial Sequence Synthetic oligonucleotide 34catgacagat ctttaggctg catatgcccc aaaccatgtc 40 35 33 DNA ArtificialSequence Description of Artificial Sequence Synthetic oligonucleotide 35catgacgtcg acatgacgac tactgcattc ctg 33 36 36 DNA Artificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 36catgacagat cttcaacgtc ccgaggtcat ttgttc 36 37 36 DNA Artificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 37catgacgcgg ccgctatgac gactactgca ttcctg 36 38 35 DNA Artificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 38catgacagat ctcaagcgaa gtaatcccgg tggtg 35 39 36 DNA Artificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 39actgtatcta gaatgaccac cacagctttc ctaatc 36 40 39 DNA Artificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 40actgtaagat ctttaagtat tcactccagc acccatagc 39 41 41 DNA ArtificialSequence Description of Artificial Sequence Synthetic oligonucleotide 41catgacgcgg ccgccctatg accaccacag ctttcctaat c 41 42 36 DNA ArtificialSequence Description of Artificial Sequence Synthetic oligonucleotide 42catgacagat ctttatatga actctgagaa gtagtc 36 43 39 DNA Artificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 43cataccgtcg acatgagaaa gaaattggag aaggcactg 39 44 39 DNA ArtificialSequence Description of Artificial Sequence Synthetic oligonucleotide 44cataccggat cctcatgctg catgagcacc aaaccatgc 39 45 42 DNA ArtificialSequence Description of Artificial Sequence Synthetic oligonucleotide 45catgacgcgg ccgctatgag aaagaaattg gagaaggcac tg 42 46 39 DNA ArtificialSequence Description of Artificial Sequence Synthetic oligonucleotide 46cataccagat cttcatgctg catgagcacc aaaccatgc 39 47 39 DNA ArtificialSequence Description of Artificial Sequence Synthetic oligonucleotide 47catatcgtcg acatggaata ttggaaacac acaaacagc 39 48 38 DNA ArtificialSequence Description of Artificial Sequence Synthetic oligonucleotide 48catgacgata tctagctgca gtttttcgga acttctgt 38 49 33 DNA ArtificialSequence Description of Artificial Sequence Synthetic oligonucleotide 49catactgcgg ccgctttaat tcaagagaac aat 33 50 35 DNA Artificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 50catatcgata tctagctgca gtttttcgga acttc 35 51 36 DNA Artificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 51catatcgtcg acatgatcat cacaaacaca atcata 36 52 36 DNA Artificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 52catgaccaga tcttattgtc tatttgtcag tatata 36 53 42 DNA Artificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 53catactgcgg ccgctcaaat agacataaca aaactgcaac gt 42 54 41 DNA ArtificialSequence Description of Artificial Sequence Synthetic oligonucleotide 54catatcgata tctatgcact agattgatac caacttccaa c 41 55 36 DNA ArtificialSequence Description of Artificial Sequence Synthetic oligonucleotide 55catatcgtcg acatgtggct gcagaacctg cttctc 36 56 34 DNA Artificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 56catgaccaga tcttcacttc tgggctggtt ccca 34 57 39 DNA Artificial SequenceDescription of Artificial Sequence Primer 57 aattttggat cctcagattccacgattttt attagaagc 39 58 34 DNA Artificial Sequence Description ofArtificial Sequence Primer 58 aaattttgtc gacatggctc ttagcaaggt caaa 3459 35 DNA Artificial Sequence Description of Artificial Sequence Primer59 ttaaggatcc tcacagttcc acatcattgt ctttg 35

What is claimed is:
 1. A prime-boost vaccination method against a bovinepathogen, comprising administering to a bovine a first, priming vaccineor immunogenic or immunological composition against the bovine pathogen,wherein the first, priming vaccine or immunogenic or immunologicalcomposition comprises a DNA vaccine or immunological or immunogeniccomposition comprising nucleic acid molecule(s) encoding and expressingin vivo in the bovine at least one immunogen from the bovine pathogen,and thereafter administering a second, boosting vaccine or immunologicalor immunogenic composition against the bovine pathogen that is differentthan the first, priming vaccine or immunological or immunogeniccomposition, but contains or expresses at least one immunogen of thebovine pathogen which is the same immunogen of the bovine pathogenexpressed by the first, priming vaccine or immunological or immunogeniccomposition.
 2. The method according to claim 1, wherein the second,boosting immunological, immunogenic or vaccine composition comprises arecombinant live viral vector that contains and expresses in vivonucleic acid molecule(s) encoding at least one immunogen of the bovinepathogen that is the same immunogen of the bovine pathogen expressed bythe first, priming vaccine or immunological or immunogenic composition.3. The method of claim 2, wherein the viral vector is a virus which is apoxvirus.
 4. The method of claim 3, wherein the poxvirus is a canarypoxvirus.
 5. The method according to claim 1, wherein the second, boostingimmunological, immunogenic or vaccine composition comprises the bovinepathogen in inactivated or attenuated form.
 6. The method according toclaim 5, wherein the second, boosting immunological, immunogenic orvaccine composition comprises the bovine pathogen in inactivated form.7. The method according to claim 1, wherein the second, boostingimmunological, immunogenic or vaccine composition comprises theimmunogen of the bovine pathogen expressed by the first, priming vaccineor immunological or immunogenic composition, as an isolated immunogen orsubunit.
 8. The method according to any one of claims 1-7 wherein thebovine pathogen is bovine respiratory syncytal virus (BRSV), bovineparainfluenza virus type-3 (bPI-3), bovine herpesvirus type-i (BHV-1) orbovine viral diarrhea virus (BVDV), or combinations thereof.
 9. Themethod of claim 8 wherein the bovine pathogen is BRSV.
 10. The method ofclaim 8 wherein the bovine pathogen is bPI-3.
 11. The method of claim 8wherein the bovine pathogen is BHV-1.
 12. The method of claim 8 whereinthe bovine pathogen is BVDV.
 13. The method of claim 12 wherein the BVDVis BVDV-1.
 14. The method of claim 12 wherein the BVDV is BVDV-2. 15.The method according to claim 8 wherein the immunogen is BRSV F, BRSV N,BRSV G, bPI-3 HN, bPI-3 F, BHV-1 gB, BHV-1 gC, BHV-1 gD, BVDV-1 E0,BVDV-1 E2, BVDV-2 E0, BVDV-2 E2, or combinations thereof.
 16. The methodaccording to claim 15 wherein the immunogen is BRSV F, BRSV N, BRSV G,or combinations thereof.
 17. The method according to claim 15 whereinthe immunogen is bPI-3 HN, bPI-3 F, or combinations thereof.
 18. Themethod according to claim 15 wherein the immunogen is BHV-1 gB, BHV-1gC, BHV-1 gD, or combinations thereof.
 19. The method according to claim15 wherein the immunogen is BVDV-1 E0, BVDV-1 E2, BVDV-2 E0, BVDV-2 E2,or combinations thereof.
 20. The method according to claim 8 wherein theimmunogen is BRSV F, BRSV N, BRSV G, bPI-3 HN, bPI-3 F, BHV-1 gB, BHV-1gC, BHV-1 gD, BVDV-1 E0, BVDV-1 E2, BVDV-2 E0, BVDV-2 E2, or epitopesthereof, or combinations thereof.
 21. The method according to any one ofclaims 1-7 wherein the bovine is a calve that can have maternalantibodies against the bovine pathogen.
 22. The method according toclaim 8 wherein the bovine is a calve that can have maternal antibodiesagainst the bovine pathogen.
 23. The method according to claim 1,wherein the DNA vaccine is administered to the bovine from calving up toand including 12 weeks of age.
 24. The method of claim 23 wherein theDNA vaccine is administered from calving up to and including 6 weeks ofage.
 25. The method of claim 23 wherein the DNA vaccine is administeredfrom calving up to and including 4 weeks of age.
 26. The method of claim23 wherein the DNA vaccine is administered from calving up to andincluding 3 weeks of age.
 27. The method according to claim 1, whereinthe second, boosting vaccine or immunological or immunogenic compositionis administered from about 2 weeks to about 5 months after the first,priming vaccine or immunological or immunogenic composition.
 28. Themethod according to claim 27, wherein the second, boosting vaccine orimmunological or immunogenic composition is administered from about 3weeks to about 6 weeks after the first, priming vaccine or immunologicalor immunogenic composition.
 29. The method according to claim 27,wherein the second, boosting vaccine or immunological or immunogeniccomposition is administered from about 4 weeks after the first, primingvaccine or immunological or immunogenic composition.
 30. The methodaccording to claim 1, including at least one additional administrationof the second, boosting vaccine or immunological or immunogeniccomposition.
 31. A kit for performing the method of claim 1 comprising(a) the first, priming vaccine or immunogenic or immunologicalcomposition, (b) the second, boosting vaccine or immunogenic orimmunological composition; wherein (a) and (b) are in separatecontainers, optionally with instructions for administration or use. 32.The kit of claim 32, wherein (a) and (b) are in separate containers inthe same package.